Transgenic plants with enhanced agronomic traits

ABSTRACT

This invention provides transgenic plant cells with recombinant DNA for expression of proteins that are useful for imparting enhanced agronomic trait(s) to transgenic crop plants. This invention also provides transgenic plants and progeny seed comprising the transgenic plant cells where the plants are selected for having an enhanced trait selected from the group of traits consisting of enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil. Also disclosed are methods for manufacturing transgenic seed and plants with enhanced traits.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/311,940, filed Dec. 19, 2005, which claims benefit under 35 USC§119(e) of U.S. provisional application Ser. No. 60/638,099, filed Dec.21, 2004, and U.S. provisional application Ser. No. 60/660,320, filedMar. 10, 2005, both of which herein incorporated by reference.

INCORPORATION OF SEQUENCE LISTING

Two copies of the sequence listing (Copy 1 and Copy 2) and a computerreadable form (CRF) of the sequence listing, all on CD-ROMs, eachcontaining the text of the file named “3126011US2.txt”, which is34,689,024 bytes (measured in MS-WINDOWS) and was created on Sep. 5,2014, are herein incorporated by reference.

INCORPORATION OF COMPUTER PROGRAM LISTING

Two copies of the Computer Program Listing (Copy 1 and Copy 2)containing folders hmmer-2.3.2 and 67pfamDir, all on CD-Rs, areincorporated herein by reference in their entirety. Folder hmmer-2.3.2contains the source code and other associated files for implementing theHMMer software for Pfam analysis. Folder 67pfamDir contains 67 PfamHidden Markov Models. Both folders were created on the CD-R on Sep. 8,2014, having a total size of 10,981,339 bytes (measured in MS-WINDOWS).

FIELD OF THE INVENTION

Disclosed herein are inventions in the field of plant genetics anddevelopmental biology. More specifically, the present inventions provideplant cells with recombinant DNA for providing an enhanced trait in atransgenic plant, plants comprising such cells, seed and pollen derivedfrom such plants, methods of making and using such cells, plants, seedsand pollen.

BACKGROUND OF THE INVENTION

Transgenic plants with improved agronomic traits such as yield,environmental stress tolerance, pest resistance, herbicide tolerance,improved seed compositions, and the like are desired by both farmers andconsumers. Although considerable efforts in plant breeding have providedsignificant gains in desired traits, the ability to introduce specificDNA into plant genomes provides further opportunities for generation ofplants with improved and/or unique traits. Merely introducingrecombinant DNA into a plant genome doesn't always produce a transgenicplant with an enhanced agronomic trait. Methods to select individualtransgenic events from a population are required to identify thosetransgenic events that are characterized by the enhanced agronomictrait.

SUMMARY OF THE INVENTION

This invention employs recombinant DNA for expression of proteins thatare useful for imparting enhanced agronomic traits to the transgenicplants. Recombinant DNA in this invention is provided in a constructcomprising a promoter that is functional in plant cells and that isoperably linked to DNA that encodes a protein having at least one aminoacid domain in a sequence that exceeds the Pfam gathering cutoff foramino acid sequence alignment with a protein domain family identified bya Pfam name in the group of Pfam names as identified in Table 28. Inmore specific embodiments of the invention the protein expressed inplant cells has an amino acid sequence with at least 90% identity to aconsensus amino acid sequence in the group of consensus amino acidsequences consisting of the consensus amino acid sequence constructedfor SEQ ID NO:84 and homologs thereof listed in Table 2 through theconsensus amino acid sequence constructed for SEQ ID NO:166 and homologsthereof listed in Table 2. In even more specific embodiments of theinvention the protein expressed in plant cells is a protein selectedfrom the group of proteins identified in Table 1.

Other aspects of the invention are specifically directed to transgenicplant cells comprising the recombinant DNA of the invention, transgenicplants comprising a plurality of such plant cells, progeny transgenicseed and transgenic pollen from such plants. Such plant cells areselected from a population of transgenic plants regenerated from plantcells transformed with recombinant DNA and that express the protein byscreening transgenic plants in the population for an enhanced trait ascompared to control plants that do not have said recombinant DNA, wherethe enhanced trait is selected from group of enhanced traits consistingof enhanced water use efficiency, enhanced cold tolerance, increasedyield, enhanced nitrogen use efficiency, enhanced seed protein andenhanced seed oil.

In yet another aspect of the invention the plant cells, plants, seedsand pollen further comprise DNA expressing a protein that providestolerance from exposure to an herbicide applied at levels that arelethal to a wild type of said plant cell. Such tolerance is especiallyuseful not only as a advantageous trait in such plants but is alsouseful in a selection step in the methods of the invention. In aspectsof the invention the agent of such herbicide is a glyphosate, dicamba,or glufosinate compound.

Yet other aspects of the invention provide transgenic plants which arehomozygous for the recombinant DNA and transgenic seed of the inventionfrom corn, soybean, cotton, canola, alfalfa, wheat or rice plants. Inother important embodiments for practice of various aspects of theinvention in Argentina the recombinant DNA is provided in plant cellsderived from corn lines that that are and maintain resistance to the Malde Rio Cuarto virus or the Puccina sorghi fungus or both.

This invention also provides methods for manufacturing non-natural,transgenic seed that can be used to produce a crop of transgenic plantswith an enhanced trait resulting from expression of stably-integrated,recombinant DNA for expressing a protein having at least one domain ofamino acids in a sequence that exceeds the Pfam gathering cutoff foramino acid sequence alignment with a protein domain family identified bya Pfam name in the group of Pfam names identified in Table 28. Morespecifically the method comprises (a) screening a population of plantsfor an enhanced trait and a recombinant DNA, where individual plants inthe population can exhibit the trait at a level less than, essentiallythe same as or greater than the level that the trait is exhibited incontrol plants which do not express the recombinant DNA, (b) selectingfrom the population one or more plants that exhibit the trait at a levelgreater than the level that said trait is exhibited in control plants,(c) verifying that the recombinant DNA is stably integrated in saidselected plants, (d) analyzing tissue of a selected plant to determinethe production of a protein having the function of a protein encoded bynucleotides in a sequence of one of SEQ ID NO:1-83; and (e) collectingseed from a selected plant. In one aspect of the invention the plants inthe population further comprise DNA expressing a protein that providestolerance to exposure to an herbicide applied at levels that are lethalto wild type plant cells and the selecting is effected by treating thepopulation with the herbicide, e.g. a glyphosate, dicamba, orglufosinate compound. In another aspect of the invention the plants areselected by identifying plants with the enhanced trait. The methods areespecially useful for manufacturing corn, soybean, cotton, alfalfa,wheat or rice seed.

Another aspect of the invention provides a method of producing hybridcorn seed comprising acquiring hybrid corn seed from a herbicidetolerant corn plant which also has stably-integrated, recombinant DNAcomprising a promoter that is (a) functional in plant cells and (b) isoperably linked to DNA that encodes a protein having at least one domainof amino acids in a sequence that exceeds the Pfam gathering cutoff foramino acid sequence alignment with a protein domain family identified bya Pfam name in the group of Pfam names identified in Table 28. Themethods further comprise producing corn plants from said hybrid cornseed, wherein a fraction of the plants produced from said hybrid cornseed is homozygous for said recombinant DNA, a fraction of the plantsproduced from said hybrid corn seed is hemizygous for said recombinantDNA, and a fraction of the plants produced from said hybrid corn seedhas none of said recombinant DNA; selecting corn plants which arehomozygous and hemizygous for said recombinant DNA by treating with anherbicide; collecting seed from herbicide-treated-surviving corn plantsand planting said seed to produce further progeny corn plants; repeatingthe selecting and collecting steps at least once to produce an inbredcorn line; and crossing the inbred corn line with a second corn line toproduce hybrid seed.

Another aspect of the invention provides a method of selecting a plantcomprising plant cells of the invention by using an immunoreactiveantibody to detect the presence of protein expressed by recombinant-DNAin seed or plant tissue. Yet another aspect of the invention providesanti-counterfeit milled seed having, as an indication of origin, a plantcells of this invention.

Still other aspects of this invention relate to transgenic plants withenhanced water use efficiency or enhanced nitrogen use efficiency. Forinstance, this invention provides methods of growing a corn, cotton orsoybean crop without irrigation water comprising planting seed havingplant cells of the invention which are selected for enhanced water useefficiency. Alternatively methods comprise applying reduced irrigationwater, e.g. providing up to 300 millimeters of ground water during theproduction of a corn crop. This invention also provides methods ofgrowing a corn, cotton or soybean crop without added nitrogen fertilizercomprising planting seed having plant cells of the invention which areselected for enhanced nitrogen use efficiency.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a DNA sequence.

FIGS. 2 and 3 are alignments of amino acid sequence.

DETAILED DESCRIPTION OF THE INVENTION

As used herein a “plant cell” means a plant cell that is transformedwith stably-integrated, non-natural, recombinant DNA, e.g. byAgrobacterium-mediated transformation or by baombardment usingmicroparticles coated with recombinant DNA or other means. A plant cellof this invention can be an originally-transformed plant cell thatexists as a microorganism or as a progeny plant cell that is regeneratedinto differentiated tissue, e.g. into a transgenic plant withstably-integrated, non-natural recombinant DNA, or seed or pollenderived from a progeny transgenic plant.

As used herein a “transgenic plant” means a plant whose genome has beenaltered by the stable integration of recombinant DNA. A transgenic plantincludes a plant regenerated from an originally-transformed plant celland progeny transgenic plants from later generations or crosses of atransformed plant.

As used herein “recombinant DNA” means DNA which has been a geneticallyengineered and constructed outside of a cell including DNA containingnaturally occurring DNA or cDNA or synthetic DNA.

As used herein “consensus sequence” means an artificial sequence ofamino acids in a conserved region of an alignment of amino acidsequences of homologous proteins, e.g. as determined by a CLUSTALWalignment of amino acid sequence of homolog proteins.

As used herein “homolog” means a protein in a group of proteins thatperform the same biological function, e.g. proteins that belong to thesame Pfam protein family and that provide a common enhanced trait intransgenic plants of this invention. Homologs are expressed byhomologous genes. Homologous genes include naturally occurring allelesand artificially-created variants. Degeneracy of the genetic codeprovides the possibility to substitute at least one base of the proteinencoding sequence of a gene with a different base without causing theamino acid sequence of the polypeptide produced from the gene to bechanged. Hence, a polynucleotide useful in the present invention mayhave any base sequence that has been changed from SEQ ID NO:1 throughSEQ ID NO:83 by substitution in accordance with degeneracy of thegenetic code. Homologs are proteins that, when optimally aligned, haveat least 60% identity, more preferably about 70% or higher, morepreferably at least 80% and even more preferably at least 90% identityover the full length of a protein identified as being associated withimparting an enhanced trait when expressed in plant cells. Homologsinclude proteins with an amino acid sequence that has at least 90%identity to a consensus amino acid sequence of proteins and homologsdisclosed herein.

Homologs are be identified by comparison of amino acid sequence, e.g.manually or by use of a computer-based tool using known homology-basedsearch algorithms such as those commonly known and referred to as BLAST,FASTA, and Smith-Waterman. A local sequence alignment program, e.g.BLAST, can be used to search a database of sequences to find similarsequences, and the summary Expectation value (E-value) used to measurethe sequence base similarity. As a protein hit with the best E-value fora particular organism may not necessarily be an ortholog or the onlyortholog, a reciprocal query is used in the present invention to filterhit sequences with significant E-values for ortholog identification. Thereciprocal query entails search of the significant hits against adatabase of amino acid sequences from the base organism that are similarto the sequence of the query protein. A hit is a likely ortholog, whenthe reciprocal query's best hit is the query protein itself or a proteinencoded by a duplicated gene after speciation. A further aspect of theinvention comprises functional homolog proteins that differ in one ormore amino acids from those of disclosed protein as the result ofconservative amino acid substitutions, for example substitutions areamong: acidic (negatively charged) amino acids such as aspartic acid andglutamic acid; basic (positively charged) amino acids such as arginine,histidine, and lysine; neutral polar amino acids such as glycine,serine, threonine, cysteine, tyrosine, asparagine, and glutamine;neutral nonpolar (hydrophobic) amino acids such as alanine, leucine,isoleucine, valine, proline, phenylalanine, tryptophan, and methionine;amino acids having aliphatic side chains such as glycine, alanine,valine, leucine, and isoleucine; amino acids having aliphatic-hydroxylside chains such as serine and threonine; amino acids havingamide-containing side chains such as asparagine and glutamine; aminoacids having aromatic side chains such as phenylalanine, tyrosine, andtryptophan; amino acids having basic side chains such as lysine,arginine, and histidine; amino acids having sulfur-containing sidechains such as cysteine and methionine; naturally conservative aminoacids such as valine-leucine, valine-isoleucine, phenylalanine-tyrosine,lysine-arginine, alanine-valine, aspartic acid-glutamic acid, andasparagine-glutamine. A further aspect of the homologs encoded by DNAuseful in the transgenic plants of the invention are those proteins thatdiffer from a disclosed protein as the result of deletion or insertionof one or more amino acids in a native sequence.

As used herein, “percent identity” means the extent to which twooptimally aligned DNA or protein segments are invariant throughout awindow of alignment of components, for example nucleotide sequence oramino acid sequence. An “identity fraction” for aligned segments of atest sequence and a reference sequence is the number of identicalcomponents that are shared by sequences of the two aligned segmentsdivided by the total number of sequence components in the referencesegment over a window of alignment which is the smaller of the full testsequence or the full reference sequence. “Percent identity” (“%identity”) is the identity fraction times 100.

As used herein “Pfam” refers to a large collection of multiple sequencealignments and hidden Markov models covering many common proteinfamilies, e.g. Pfam version 18.0 (August 2005) contains alignments andmodels for 7973 protein families and is based on the Swissprot 47.0 andSP-TrEMBL 30.0 protein sequence databases. See S. R. Eddy, “ProfileHidden Markov Models”, Bioinformatics 14:755-763, 1998. Pfam iscurrently maintained and updated by a Pfam Consortium. The alignmentsrepresent some evolutionary conserved structure that has implicationsfor the protein's function. Profile hidden Markov models (profile HMMs)built from the Pfam alignments are useful for automatically recognizingthat a new protein belongs to an existing protein family even if thehomology by alignment appears to be low. Once one DNA is identified asencoding a protein which imparts an enhanced trait when expressed intransgenic plants, other DNA encoding proteins in the same proteinfamily are identified by querying the amino acid sequence of proteinencoded by candidate DNA against the Hidden Markov Model whichcharacterizes the Pfam domain using HMMER software, a current version ofwhich is provided in the appended computer listing. Candidate proteinsmeeting the gathering cutoff for the alignment of a particular Pfam arein the protein family and have cognate DNA that is useful inconstructing recombinant DNA for the use in the plant cells of thisinvention. Hidden Markov Model databases for use with HMMER software inidentifying DNA expressing protein in a common Pfam for recombinant DNAin the plant cells of this invention are also included in the appendedcomputer listing. The HMMER software and Pfam databases are version 18.0and were used to identify known domains in the proteins corresponding toamino acid sequence of SEQ ID NO:84 through SEQ ID NO:166. All DNAencoding proteins that have scores higher than the gathering cutoffdisclosed in Table 27 by Pfam analysis disclosed herein can be used inrecombinant DNA of the plant cells of this invention, e.g. for selectingtransgenic plants having enhanced agronomic traits. The relevant Pfamsfor use in this invention, as more specifically disclosed below, areAAA, AP2, Aldo ket red, Alpha-amylase, Aminotran 1 2, Ank, ArfGap, Asnsynthase, BRO1, CBFD NFYB HMF, Catalase, CorA, Cpn60 TCP1, Cystatin, DNAphotolyase, DSPc, DUF1685, DUF296, Di19, E2F TDP, FAD binding 7, FAdesaturase, FBPase, GAF, GATA, GATase 2, Glyco hydro 1, Glyoxalase,Got1, HATPase c, HSF DNA-bind, HSP20, HisKA, Homeobox, Hpt, IsoamylaseN, K-box, Lactamase B, Metallophos, MtN3 slv, NAF, NAM, NIF, OxidoredFMN, PAS, PDZ, PRA1, Peptidase C15, Peptidase S10, Peptidase S41,Phytochrome, Peinase, Pkinase Tyr, Pyridoxal deC, RIO1, RRM 1, RTC, RTCinsert, Ras, Response reg, SPC25, SPX, SRF-TF, Synaptobrevin, UPF0057,zf-C2H2, and zf-C3HC4, the databases for which are included in theappended computer listing.

As used herein “promoter” means regulatory DNA for initializingtranscription. A “plant promoter” is a promoter capable of initiatingtranscription in plant cells whether or not its origin is a plant cell,e.g. is it well known that Agrobacterium promoters are functional inplant cells. Thus, plant promoters include promoter DNA obtained fromplants, plant viruses and bacteria such as Agrobacterium andBradyrhizobium bacteria. Examples of promoters under developmentalcontrol include promoters that preferentially initiate transcription incertain tissues, such as leaves, roots, or seeds. Such promoters arereferred to as “tissue preferred”. Promoters that initiate transcriptiononly in certain tissues are referred to as “tissue specific”. A “celltype” specific promoter primarily drives expression in certain celltypes in one or more organs, for example, vascular cells in roots orleaves. An “inducible” or “repressible” promoter is a promoter which isunder environmental control. Examples of environmental conditions thatmay effect transcription by inducible promoters include anaerobicconditions, or certain chemicals, or the presence of light. Tissuespecific, tissue preferred, cell type specific, and inducible promotersconstitute the class of “non-constitutive” promoters. A “constitutive”promoter is a promoter which is active under most conditions.

As used herein “operably linked” means the association of two or moreDNA fragments in a DNA construct so that the function of one, e.g.protein-encoding DNA, is controlled by the other, e.g. a promoter.

As used herein “expressed” means produced, e.g. a protein is expressedin a plant cell when its cognate DNA is transcribed to mRNA that istranslated to the protein.

As used herein a “control plant” means a plant that does not contain therecombinant DNA that expressed a protein that impart an enhanced trait.A control plant is to identify and select a transgenic plant that has anenhance trait. A suitable control plant can be a non-transgenic plant ofthe parental line used to generate a transgenic plant, i.e. devoid ofrecombinant DNA. A suitable control plant may in some cases be a progenyof a hemizygous transgenic plant line that is does not contain therecombinant DNA, known as a negative sergeant.

As used herein an “enhanced trait” means a characteristic of atransgenic plant that includes, but is not limited to, an enhanceagronomic trait characterized by enhanced plant morphology, physiology,growth and development, yield, nutritional enhancement, disease or pestresistance, or environmental or chemical tolerance. In more specificaspects of this invention enhanced trait is selected from group ofenhanced traits consisting of enhanced water use efficiency, enhancedcold tolerance, increased yield, enhanced nitrogen use efficiency,enhanced seed protein and enhanced seed oil. In an important aspect ofthe invention the enhanced trait is enhanced yield including increasedyield under non-stress conditions and increased yield underenvironmental stress conditions. Stress conditions may include, forexample, drought, shade, fungal disease, viral disease, bacterialdisease, insect infestation, nematode infestation, cold temperatureexposure, heat exposure, osmotic stress, reduced nitrogen nutrientavailability, reduced phosphorus nutrient availability and high plantdensity. “Yield” can be affected by many properties including withoutlimitation, plant height, pod number, pod position on the plant, numberof intemodes, incidence of pod shatter, grain size, efficiency ofnodulation and nitrogen fixation, efficiency of nutrient assimilation,resistance to biotic and abiotic stress, carbon assimilation, plantarchitecture, resistance to lodging, percent seed germination, seedlingvigor, and juvenile traits. Yield can also affected by efficiency ofgermination (including germination in stressed conditions), growth rate(including growth rate in stressed conditions), ear number, seed numberper ear, seed size, composition of seed (starch, oil, protein) andcharacteristics of seed fill.

Increased yield of a transgenic plant of the present invention can bemeasured in a number of ways, including test weight, seed number perplant, seed weight, seed number per unit area (i.e. seeds, or weight ofseeds, per acre), bushels per acre, tonnes per acre, tons per acre, kiloper hectare. For example, maize yield may be measured as production ofshelled corn kernels per unit of production area, for example in bushelsper acre or metric tons per hectare, often reported on a moistureadjusted basis, for example at 15.5 percent moisture. Increased yieldmay result from improved utilization of key biochemical compounds, suchas nitrogen, phosphorous and carbohydrate, or from improved responses toenvironmental stresses, such as cold, heat, drought, salt, and attack bypests or pathogens. Recombinant DNA used in this invention can also beused to provide plants having improved growth and development, andultimately increased yield, as the result of modified expression ofplant growth regulators or modification of cell cycle or photosynthesispathways. Also of interest is the generation of transgenic plants thatdemonstrate enhanced yield with respect to a seed component that may ormay not correspond to an increase in overall plant yield. Suchproperties include enhancements in seed oil, seed molecules such astocopherol, protein and starch, or oil particular oil components as maybe manifest by an alterations in the ratios of seed components.

A subset of the nucleic molecules of this invention includes fragmentsof the disclosed recombinant DNA consisting of oligonucleotides of atleast 15, preferably at least 16 or 17, more preferably at least 18 or19, and even more preferably at least 20 or more, consecutivenucleotides. Such oligonucleotides are fragments of the larger moleculeshaving a sequence selected from the group consisting of SEQ ID NO:1through SEQ ID NO:83, and find use, for example as probes and primersfor detection of the polynucleotides of the present invention.

In some embodiments of the present invention, a dominant negative mutantof a native gene is generated to achieve the desired effect. As usedherein, “dominant negative mutant” means a mutant gene whose geneproduct adversely affects the normal, wild-type gene product within thesame cell, usually by dimerizing (combining) with it. In cases ofpolymeric molecules, such as collagen, dominant negative mutations areoften more deleterious than mutations causing the production of no geneproduct (null mutations or null alleles). SEQ ID NO: 6 and SEQ ID NO: 7are constructed to encode agl11 protein with K-box deleted and MADs 3protein with MAD box deleted, respectively. MADS box proteins similar toAGL11 can be considered as having three functional domains. There is anN-terminal DNA-binding domain (the MADS box), a more distal dimerizationdomain (the K-box) and a C-terminal domain that is usually involved ininteractions with other proteins. In plants the region between the MADSbox and the K-box has been shown to be important for DNA binding in someproteins and is often referred to as the I-box (Fan et al., 1997).Several different classes of dominant negative constructs areconsidered. Deletion or inactivation of the DNA-binding domain cancreate proteins that are able to dimerize with their native full lengthcounterparts as well as other natural dimerization partners. Likewise,removal of the C-terminal domain can allow dimerization with both thenative protein and it's natural dimerization partners. In both casesthese types of constructs disable both the target protein and any otherprotein capable of interacting with the K-box.

In other embodiments of the invention a constitutively active mutant isconstructed to achieve the desired effect. SEQ ID NO:3 encodes only thekinase domain from a calcium-dependent protein kinase (CDPK). CDPK1 hasa domain structure similar to other calcium-dependant protein kinases inwhich the protein kinase domain is separated from four efhand domains by42 amino acid “spacer” region. Calcium-dependant protein kinases arethought to be activated by a calcium-induced conformational change thatresults in movement of an autoinhibitory domain away from the proteinkinase active site (Yokokura et al., 1995). Thus, constitutively activeproteins can be made by over expressing the protein kinase domain alone.

DNA constructs are assembled using methods well known to persons ofordinary skill in the art and typically comprise a promoter operablylinked to DNA, the expression of which provides the enhanced agronomictrait. Other construct components may include additional regulatoryelements, such as 5′ leasders and introns for enhancing transcription,3′ untranslated regions (such as polyadenylation signals and sites), DNAfor transit or signal peptides.

Numerous promoters that are active in plant cells have been described inthe literature. These include promoters present in plant genomes as wellas promoters from other sources, including nopaline synthase (NOS)promoter and octopine synthase (OCS) promoters carried on tumor-inducingplasmids of Agrobacterium tumefaciens, caulimovirus promoters such asthe cauliflower mosaic virus. For instance, see U.S. Pat. Nos. 5,858,742and 5,322,938, which disclose versions of the constitutive promoterderived from cauliflower mosaic virus (CaMV35S), U.S. Pat. No.5,641,876, which discloses a rice actin promoter, U.S. PatentApplication Publication 2002/0192813A1, which discloses 5′, 3′ andintron elements useful in the design of effective plant expressionvectors, U.S. patent application Ser. No. 09/757,089, which discloses amaize chloroplast aldolase promoter, U.S. patent application Ser. No.08/706,946, which discloses a rice glutelin promoter, U.S. patentapplication Ser. No. 09/757,089, which discloses a maize aldolase (FDA)promoter, and U.S. patent application Ser. No. 60/310, 370, whichdiscloses a maize nicotianamine synthase promoter, all of which areincorporated herein by reference. These and numerous other promotersthat function in plant cells are known to those skilled in the art andavailable for use in recombinant polynucleotides of the presentinvention to provide for expression of desired genes in transgenic plantcells.

In some aspects of the invention, sufficient expression in plant seedtissues is desired to effect improvements in seed composition. Exemplarypromoters for use for seed composition modification include promotersfrom seed genes such as napin (U.S. Pat. No. 5,420,034), zein Z27 andglutelin1 (Russell et al. (1997) Transgenic Res. 6(2):157-166), andperoxiredoxin antioxidant (Per1) (Stacy et al. (1996) Plant Mol Biol.31(6):1205-1216), maize L3 oleosin (U.S. Pat. No. 6,433,252), globulin 1(Belanger et al (1991) Genetics 129:863-872).

In other aspects of the invention, preferential expression in plantgreen tissues is desired. Promoters of interest for such uses includethose from genes such as Arabidopsis thaliana ribulose-1,5-bisphosphatecarboxylase (Rubisco) small subunit (Fischhoff et al. (1992) Plant MolBiol. 20:81-93), aldolase and pyruvate orthophosphate dikinase (PPDK)(Taniguchi et al. (2000) Plant Cell Physiol. 41(1):42-48).

Furthermore, the promoters may be altered to contain multiple “enhancersequences” to assist in elevating gene expression. Such enhancers areknown in the art. By including an enhancer sequence with suchconstructs, the expression of the selected protein may be enhanced.These enhancers often are found 5′ to the start of transcription in apromoter that functions in eukaryotic cells, but can often be insertedupstream (5′) or downstream (3′) to the coding sequence. In someinstances, these 5′ enhancing elements are introns. Particularly usefulas enhancers are the 5′ introns of the rice actin 1 (see U.S. Pat. No.5,641,876) and rice actin 2 genes, the maize alcohol dehydrogenase geneintron, the maize heat shock protein 70 gene intron (U.S. Pat. No.5,593,874) and the maize shrunken 1 gene.

In other aspects of the invention, sufficient expression in plant seedtissues is desired to effect improvements in seed composition. Exemplarypromoters for use for seed composition modification include promotersfrom seed genes such as napin (U.S. Pat. No. 5,420,034), maize L3oleosin (U.S. Pat. No. 6,433,252), zein Z27 (Russell et al. (1997)Transgenic Res. 6(2):157-166), globulin 1 (Belanger et al (1991)Genetics 129:863-872), glutelin 1 (Russell (1997) supra), andperoxiredoxin antioxidant (Per1) (Stacy et al. (1996) Plant Mol Biol.31(6):1205-1216).

Recombinant DNA constructs prepared in accordance with the inventionwill also generally include a 3′ element that typically contains apolyadenylation signal and site. Well-known 3′ elements include thosefrom Agrobacterium tumefaciens genes such as nos 3′, tml 3′, tmr 3′, tms3′, ocs 3′, tr7 3′, for example disclosed in U.S. Pat. No. 6,090,627,incorporated herein by reference; 3′ elements from plant genes such aswheat (Triticum aesevitum) heat shock protein 17 (Hsp17 3′), a wheatubiquitin gene, a wheat fructose-1,6-biphosphatase gene, a rice glutelingene a rice lactate dehydrogenase gene and a rice beta-tubulin gene, allof which are disclosed in U.S. published patent application 2002/0192813A1, incorporated herein by reference; and the pea (Pisum sativum)ribulose biphosphate carboxylase gene (rbs 3), and 3′ elements from thegenes within the host plant.

Constructs and vectors may also include a transit peptide for targetingof a gene target to a plant organelle, particularly to a chloroplast,leucoplast or other plastid organelle. For descriptions of the use ofchloroplast transit peptides see U.S. Pat. No. 5,188,642 and U.S. Pat.No. 5,728,925, incorporated herein by reference. For description of thetransit peptide region of an Arabidopsis EPSPS gene useful in thepresent invention, see Klee, H. J. et al (MGG (1987) 210:437-442).

Transgenic plants comprising or derived from plant cells of thisinvention transformed with recombinant DNA can be further enhanced withstacked traits, e.g. a crop plant having an enhanced trait resultingfrom expression of DNA disclosed herein in combination with herbicideand/or pest resistance traits. For example, genes of the currentinvention can be stacked with other traits of agronomic interest, suchas a trait providing herbicide resistance, or insect resistance, such asusing a gene from Bacillus thuringensis to provide resistance againstlepidopteran, coliopteran, homopteran, hemiopteran, and other insects.Herbicides for which transgenic plant tolerance has been demonstratedand the method of the present invention can be applied include, but arenot limited to, glyphosate, dicamba, glufosinate, sulfonylurea,bromoxynil and norflurazon herbicides. Polynucleotide molecules encodingproteins involved in herbicide tolerance are well-known in the art andinclude, but are not limited to, a polynucleotide molecule encoding5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) disclosed in U.S.Pat. Nos. 5,094,945; 5,627,061; 5,633,435 and 6,040,497 for impartingglyphosate tolerance; polynucleotide molecules encoding a glyphosateoxidoreductase (GOX) disclosed in U.S. Pat. No. 5,463,175 and aglyphosate-N-acetyl transferase (GAT) disclosed in U.S. PatentApplication publication 2003/0083480 A1 also for imparting glyphosatetolerance; dicamba monooxygenase disclosed in U.S. Patent Applicationpublication 2003/0135879 A1 for imparting dicamba tolerance; apolynucleotide molecule encoding bromoxynil nitrilase (Bin) disclosed inU.S. Pat. No. 4,810,648 for imparting bromoxynil tolerance; apolynucleotide molecule encoding phytoene desaturase (crtl) described inMisawa et al, (1993) Plant J. 4:833-840 and Misawa et al, (1994) PlantJ. 6:481-489 for norflurazon tolerance; a polynucleotide moleculeencoding acetohydroxyacid synthase (AHAS, aka ALS) described inSathasiivan et al. (1990) Nucl. Acids Res. 18:2188-2193 for impartingtolerance to sulfonylurea herbicides; polynucleotide molecules known asbar genes disclosed in DeBlock, et al. (1987) EMBO J. 6:2513-2519 forimparting glufosinate and bialaphos tolerance; polynucleotide moleculesdisclosed in U.S. Patent Application Publication 2003/010609 A1 forimparting N-amino methyl phosphonic acid tolerance; polynucleotidemolecules disclosed in U.S. Pat. No. 6,107,549 for impartinig pyridineherbicide resistance; molecules and methods for imparting tolerance tomultiple herbicides such as glyphosate, atrazine, ALS inhibitors,isoxoflutole and glufosinate herbicides are disclosed in U.S. Pat. No.6,376,754 and U.S. Patent Application Publication 2002/0112260, all ofsaid U.S. patents and Patent Application Publications are incorporatedherein by reference. Molecules and methods for impartinginsect/nematode/virus resistance is disclosed in U.S. Pat. Nos.5,250,515; 5,880,275; 6,506,599; 5,986,175 and U.S. Patent ApplicationPublication 2003/0150017 A1, all of which are incorporated herein byreference.

In particular embodiments, the inventors contemplate the use ofantibodies, either monoclonal or polyclonal which bind to the proteinsdisclosed herein. Means for preparing and characterizing antibodies arewell known in the art (See, e.g., Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, 1988; incorporated herein by reference). Themethods for generating monoclonal antibodies (mAbs) generally beginalong the same lines as those for preparing polyclonal antibodies.Briefly, a polyclonal antibody is prepared by immunizing an animal withan immunogenic composition in accordance with the present invention andcollecting antisera from that immunized animal. A wide range of animalspecies can be used for the production of antisera. Typically the animalused for production of anti-antisera is a rabbit, a mouse, a rat, ahamster, a guinea pig or a goat. Because of the relatively large bloodvolume of rabbits, a rabbit is a preferred choice for production ofpolyclonal antibodies.

As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin can alsobe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include using glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

As is also well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Exemplary andpreferred adjuvants include complete Freund's adjuvant (a non-specificstimulator of the immune response containing killed Mycobacteriumtuberculosis), incomplete Freund's adjuvants and aluminum hydroxideadjuvant.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen (subcutaneous, intramuscular, intradermal, intravenous andintraperitoneal). The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization. A second, booster, injection may also be given.The process of boosting and titering is repeated until a suitable titeris achieved. When a desired level of immunogenicity is obtained, theimmunized animal can be bled and the serum isolated and stored, and/orthe animal can be used to generate mAbs.

mAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g., a purified orpartially purified antifungal protein, polypeptide or peptide. Theimmunizing composition is administered in a manner effective tostimulate antibody producing cells. Rodents such as mice and rats arepreferred animals, however, the use of rabbit, sheep, or frog cells isalso possible. The use of rats may provide certain advantages (Goding,1986, pp. 60-61), but mice are preferred, with the BALB/c mouse beingmost preferred as this is most routinely used and generally gives ahigher percentage of stable fusions.

Following immunization, somatic cells with the potential for producingantibodies, specifically B lymphocytes (B cells), are selected for usein the mAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible. Often, a panel of animals will have beenimmunized and the spleen of animal with the highest antibody titer willbe removed and the spleen lymphocytes obtained by homogenizing thespleen with a syringe. Typically, a spleen from an immunized mousecontains approximately 5×10⁷ to 2×10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render them incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, 1986, pp. 65-66; Campbell, 1984, pp.75-83). For example, where the immunized animal is a mouse, one may useP3-X63/Ag8, X63-Ag8.653, NSI/1.Ag 41, Sp210-Ag14, FO, NSO/U, MPC-11,MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3,Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 andUC729-6 are all useful in connection with human cell fusions.

One preferred murine myeloma cell is the NS-1 myeloma cell line (alsotermed P3-NS-1-Ag4-1), which is readily available from the NIGMS HumanGenetic Mutant Cell Repository by requesting cell line repository numberGM3573. Another mouse myeloma cell line that may be used is the8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cellline.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1to about 1:1, respectively, in the presence of an agent or agents(chemical or electrical) that promote the fusion of cell membranes.Fusion methods using Spend virus have been described (Kohler andMilstein, 1975; 1976), and those using polyethylene glycol (PEG), suchas 37% (v/v) PEG, (Gefter et al., 1977). The use of electrically inducedfusion methods is also appropriate (Goding, 1986, pp. 71-74).

Fusion procedures usually produce viable hybrids at low frequencies,about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azasenne blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B-cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B-cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide mAbs. The cell lines may be exploitedfor mAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide mAbs in high concentration. The individualcell lines could also be cultured in vitro, where the mAbs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations. mAbs produced by either means may be furtherpurified, if desired, using filtration, centrifugation and variouschromatographic methods such as HPLC or affinity chromatography.

Plant Cell Transformation Methods

Numerous methods for transforming plant cells with recombinant DNA areknown in the art and may be used in the present invention. Two commonlyused methods for plant transformation are Agrobacterium-mediatedtransformation and microprojectile bombardment. Microprojectilebombardment methods are illustrated in U.S. Pat. No. 5,015,580(soybean); U.S. Pat. No. 5,550,318 (corn); U.S. Pat. No. 5,538,880(corn); U.S. Pat. No. 5,914,451 (soybean); U.S. Pat. No. 6,160,208(corn); U.S. Pat. No. 6,399,861 (corn) and U.S. Pat. No. 6,153,812(wheat) and Agrobacterium-mediated transformation is described in U.S.Pat. No. 5,159,135 (cotton); U.S. Pat. No. 5,824,877 (soybean); U.S.Pat. No. 5,591,616 (corn); and U.S. Pat. No. 6,384,301 (soybean), all ofwhich are incorporated herein by reference. For Agrobacteriumtumefaciens based plant transformation system, additional elementspresent on transformation constructs will include T-DNA left and rightborder sequences to facilitate incorporation of the recombinantpolynucleotide into the plant genome.

In general it is useful to introduce recombinant DNA randomly, i.e. at anon-specific location, in the genome of a target plant line. In specialcases it may be useful to target recombinant DNA insertion in order toachieve site-specific integration, for example to replace an existinggene in the genome, to use an existing promoter in the plant genome, orto insert a recombinant polynucleotide at a predetermined site known tobe active for gene expression. Several site specific recombinationsystems exist which are known to function implants include cre-lox asdisclosed in U.S. Pat. No. 4,959,317 and FLP-FRT as disclosed in U.S.Pat. No. 5,527,695, both incorporated herein by reference.

Transformation methods of this invention are preferably practiced intissue culture on media and in a controlled environment. “Media” refersto the numerous nutrient mixtures that are used to grow cells in vitro,that is, outside of the intact living organism. Recipient cell targetsinclude, but are not limited to, meristem cells, callus, immatureembryos and gametic cells such as microspores, pollen, sperm and eggcells. It is contemplated that any cell from which a fertile plant maybe regenerated is useful as a recipient cell. Callus may be initiatedfrom tissue sources including, but not limited to, immature embryos,seedling apical meristems, microspores and the like. Cells capable ofproliferating as callus are also recipient cells for genetictransformation. Practical transformation methods and materials formaking transgenic plants of this invention, for example various mediaand recipient target cells, transformation of immature embryo cells andsubsequent regeneration of fertile transgenic plants are disclosed inU.S. Pat. Nos. 6,194,636 and 6,232,526, which are incorporated herein byreference.

The seeds of transgenic plants can be harvested from fertile transgenicplants and be used to grow progeny generations of transformed plants ofthis invention including hybrid plants line for selection of plantshaving an enhanced trait. In addition to direct transformation of aplant with a recombinant DNA, transgenic plants can be prepared bycrossing a first plant having a recombinant DNA with a second plantlacking the DNA. For example, recombinant DNA can be introduced intofirst plant line that is amenable to transformation to produce atransgenic plant which can be crossed with a second plant line tointrogress the recombinant DNA into the second plant line. A transgenicplant with recombinant DNA providing an enhanced trait, e.g. enhancedyield, can be crossed with transgenic plant line having otherrecombinant DNA that confers another trait, for example herbicideresistance or pest resistance, to produce progeny plants havingrecombinant DNA that confers both traits. Typically, in such breedingfor combining traits the transgenic plant donating the additional traitis a male line and the transgenic plant carrying the base traits is thefemale line. The progeny of this cross will segregate such that some ofthe plants will carry the DNA for both parental traits and some willcarry DNA for one parental trait; such plants can be identified bymarkers associated with parental recombinant DNA, e.g. markeridentification by analysis for recombinant DNA or, in the case where aselectable marker is linked to the recombinant, by application of theselecting agent such as a herbicide for use with a herbicide tolerancemarker, or by selection for the enhanced trait. Progeny plants carryingDNA for both parental traits can be crossed back into the female parentline multiple times, for example usually 6 to 8 generations, to producea progeny plant with substantially the same genotype as one originaltransgenic parental line but for the recombinant DNA of the othertransgenic parental line

In the practice of transformation DNA is typically introduced into onlya small percentage of target plant cells in any one transformationexperiment. Marker genes are used to provide an efficient system foridentification of those cells that are stably transformed by receivingand integrating a transgenic DNA construct into their genomes. Preferredmarker genes provide selective markers which confer resistance to aselective agent, such as an antibiotic or herbicide. Any of theherbicides to which plants of this invention may be resistant are usefulagents for selective markers. Potentially transformed cells are exposedto the selective agent. In the population of surviving cells will bethose cells where, generally, the resistance-conferring gene isintegrated and expressed at sufficient levels to permit cell survival.Cells may be tested further to confirm stable integration of theexogenous DNA. Commonly used selective marker genes include thoseconferring resistance to antibiotics such as kanamycin and paromomycin(nptII), hygromycin B (aph IV) and gentamycin (aac3 and aacC4) orresistance to herbicides such as glufosinate (bar or pat) and glyphosate(aroA or EPSPS). Examples of such selectable are illustrated in U.S.Pat. Nos. 5,550,318; 5,633,435; 5,780,708 and 6,118,047, all of whichare incorporated herein by reference. Selectable markers which providean ability to visually identify transformants can also be employed, forexample, a gene expressing a colored or fluorescent protein such as aluciferase or green fluorescent protein (GFP) or a gene expressing abeta-glucuronidase or uidA gene (GUS) for which various chromogenicsubstrates are known.

Plant cells that survive exposure to the selective agent, or plant cellsthat have been scored positive in a screening assay, may be cultured inregeneration media and allowed to mature into plants. Developingplantlets regenerated from transformed plant cells can be transferred toplant growth mix, and hardened off, for example, in an environmentallycontrolled chamber at about 85% relative humidity, 600 ppm CO₂, and25-250 microeinsteins m⁻² s⁻¹ of light, prior to transfer to agreenhouse or growth chamber for maturation. Plants are regenerated fromabout 6 weeks to 10 months after a transformant is identified, dependingon the initial tissue. Plants may be pollinated using conventional plantbreeding methods known to those of skill in the art and seed produced,for example self-pollination is commonly used with transgenic corn. Theregenerated transformed plant or its progeny seed or plants can betested for expression of the recombinant DNA and selected for thepresence of enhanced agronomic trait.

Transgenic Plants and Seeds

Transgenic plants derived from the plant cells of this invention aregrown to generate transgenic plants having an enhanced trait as comparedto a control plant and produce transgenic seed and haploid pollen ofthis invention. Such plants with enhanced traits are identified byselection of transformed plants or progeny seed for the enhanced trait.For efficiency a selection method is designed to evaluate multipletransgenic plants (events) comprising the recombinant DNA, for examplemultiple plants from 2 to 20 or more transgenic events. Transgenicplants grown from transgenic seed provided herein demonstrate improvedagronomic traits that contribute to increased yield or other trait thatprovides increased plant value, including, for example, improved seedquality. Of particular interest are plants having enhanced water useefficiency, enhanced cold tolerance, increased yield, enhanced nitrogenuse efficiency, enhanced seed protein and enhanced seed oil.

Table 1 provides a list of protein encoding DNA (“genes”) that areuseful as recombinant DNA for production of transgenic plants withenhanced agronomic trait, the elements of Table 1 are described byreference to:

“PEP SEQ” which identifies an amino acid sequence from SEQ ID NO:84 to166.“NUC SEQ” which identifies a DNA sequence from SEQ ID NO:1 to 83.“Base Vector” which identifies a base plasmid used for transformation ofthe recombinant DNA.“PROTEIN NAME” which is a common name for protein encoded by therecombinant DNA.“Enhanced trait” which identifies an enhanced trait which is imparted bythe expression of the protein in a transgenic crop plant.“Plasmid ID” which identifies an arbitrary name for the planttransformation plasmid comprising recombinant DNA for expressing therecombinant DNA in plant cells.

TABLE 1 PEP NUC SEQ SEQ ID ID NO NO Base Vector PROTEIN NAME Enhancedtrait(s) Plasmid ID 84 1 pMON65154 lactoylglutathione lyase Enhancedseed protein pMON69462 85 2 pMON72472 rab7c Enhanced cold tolerancepMON69456 86 3 pMON65154 CDPK kinase domain Enhanced water use pMON67754efficiency 87 4 pMON72472 SCOF-1 Enhanced water use pMON72494 efficiencyand enhanced cold tolerance 88 5 pMON72472 Synechococcus sp. PCCIncreased yield, enhanced pMON68399 6301 Delta9 desaturase coldtolerance and enhanced water use efficiency 89 6 pMON72472 Arabidopsisagl11 delta Improved cold tolerance pMON73765 K-box 90 7 pMON72472 riceMADS3 delta Enhanced cold tolerance pMON73829 MADS-box - L37528 91 8pMON72472 corn MADS box Enhanced nitrogen use pMON73816 protein 110efficiency and enhance cold tolerance 92 9 pMON72472 ArabidopsisEnhanced cold tolerance pMON75305 homeodomain transcription factor- 9310 pMON72472 Arabidopsis AP2 Enhanced cold tolerance pMON75306 domaintranscription factor 94 11 pMON72472 Arabidopsis GATA Enhanced coldtolerance pMON75309 domain transcription factor 95 12 pMON72472Arabidopsis AT-hook Enhanced cold tolerance pMON75312 domaintranscription factor- 96 13 pMON72472 rice DETI-like - Enhanced nitrogenuse pMON80270 BAB16336 efficiency and enhanced cold tolerance 97 14pMON72472 soybean G482-like 1 Enhanced water use pMON76342 efficiency 9815 pMON72472 Arabidopsis Enhanced cold tolerance pMON79174 hypotheticalprotein [NM_114802] 99 16 pMON72472 corn hypothetical Enhanced coldtolerance pMON79413 protein 100 17 pMON72472 soy Pra2-like protein 2Enhanced nitrogen use pMON75511 efficiency 101 18 pMON72472Agrobacterium Enhanced cold tolerance pMON75515 cryptochrome-likeprotein - AE008050 102 19 pMON72472 rice SNF1-like protein Enhancednitrogen use pMON80542 9[OsPK4] - AB011967 efficiency, enhanced wateruse efficiency, increased yield 103 20 pMON72472 corn SNF1-like proteinEnhanced water use pMON78949 3 efficiency and enhanced nitrogen useefficiency 104 21 pMON72472 corn SNF1-like protein Enhanced coldtolerance pMON78936 8 and enhanced water use efficiency 105 22 pMON72472Corn Rubisco Activase Increased yield, enhanced pMON75524 2 coldtolerance and enhanced nitrogen use efficiency 106 23 pMON72472 NLIInteracting Isoform Enhanced cold tolerance pMON79163 T1- and increasedyield 107 24 pMON72472 maize synaptobrevin- Enhanced cold tolerancepMON75533 related sequnece 1 - condition and increased yield 108 25pMON72472 maize magnesium Enhanced nitrogen use pMON79709 transportermrs2-1-like efficiency and increased 1 sequence yield 109 26 pMON72472Corn Protein similar to Enhanced water use pMON79422 nodulin MtN3protein efficiency 110 27 pMON72472 Corn glyoxalase II Enhanced coldtolerance pMON79425 isozyme 111 28 pMON72472 Corn RNA 3- Enhanced coldtolerance pMON79718 TERMINAL PHOSPHATE CYCLASE-LIKE PROTEIN 112 29pMON72472 rice Di19 like sequence Enhanced cold tolerance pMON79447 11330 pMON72472 soybean MAP kinase 6 Enhanced cold tolerance pMON78232 like2 sequence 114 31 pMON72472 Ralstonia metallidurans Enhanced coldtolerance, pMON75980 glutamate and enhanced nitrogen use decarboxylaseefficiency 115 32 pMON72472 rice HSF5 like Enhanced water use pMON80489sequence efficiency 116 33 pMON72472 soybean hsp17.4 like 1 Enhancedcold tolerance pMON79697 sequence and enhanced water use efficiency 11734 pMON72472 Corn putative Enhanced water use pMON78237 pyrrolidonecarboxyl efficiency peptidase 118 35 pMON72472 Arabidopsis E2F Enhancedcold tolerance pMON80461 enhanced nitrogen use efficiency 119 36pMON72472 Arabidopsis protein Enhanced cold tolerance pMON78235phosphatase 1A 120 37 pMON72472 Arabidopsis CtpA Enhanced coldtolerance, pMON80452 and enhanced water use efficiency 121 38 pMON74532Arabidopsis CtpA Increased yield 122 39 pMON72472 Corn protein similarto Enhanced cold tolerance pMON80500 Arabidopsis Probable microsomalsignal peptidase 123 40 pMON72472 [Oryza sativa] putative Enhancednitrogen use pMON80850 aldose reductase efficiency 124 41 pMON72472 ZeaMays Kinase II Increased seed protein pMON78949 (similar to Yeast 1KS1 &At MRK1) 125 42 pMON72472 Fructose-1-6- Increased yield pMON81853bisphosphatase 126 43 pMON72472 soy G1928 like 1 Increased seed proteinpMON83769 127 44 pMON74532 Synechocystis sp. 6803 Increased yieldpMON78911 Hik19 128 45 pMON72472 Synechocystis sp. 6803 Increased yieldHik19 129 46 pMON72472 Arabidopsis NAC Increased yield pMON73787 domaintranscription factor 130 47 pMON72472 yeast alanine Increased yield andpMON77895 aminotransferase 1 - enhanced nitrogen use AAB67593 efficiency131 48 pMON72472 soybean catalase-like 1 Increased yield pMON79152 13249 pMON72472 corn ALG-2 interacting Increased yield pMON80921 protein133 50 pMON72472 Putative Serine Increased yield pMON75505Carboxypeptidase- 134 51 pMON72472 Putative Ankyrin Like Increased yieldpMON80925 Protein- 135 52 pMON72472 Putative Kinase Like Increased yieldpMON78942 Protein- 136 53 pMON72472 Putative Protein- Increased yieldpMON79164 137 54 pMON72472 yeast YPR145W/asn1 - Increased yieldpMON79653 U40829 138 55 pMON72472 rice AtHSP17.6A like 1 Increased yieldpMON81228 sequence 139 56 pMON72472 yeast YDL123w Increased yieldpMON79430 140 57 pMON72472 rice 12- Increased yield pMON7973loxophytodienoate reductase like 1 sequence 141 58 pMON72472 soybean MAPkinase 6 Increased yield pMON78229 like 3 sequence 142 59 pMON72472Arabidopsis GAD1 Increased yield pMON79696 143 60 pMON74532 ArabidopsisGAD1 144 61 pMON72472 soybean hsp17.4 like 4 Increased yield pMON78240sequence 145 62 pMON72472 maize hsp60 like 4 Increased yield pMON80283sequence 146 63 pMON72472 soy dsPTP 1 Increased yield pMON80866 147 64pMON72472 Yeast GLC3 Glycogen Increased yield pMON80292 branching enzyme148 65 pMON72472 Arabidopsis unknown Increased yield pMON82223 protein149 66 pMON72472 beta-D-glucosidase Increased yield pMON83553 150 67pMON72472 unknown protein 1 Increased yield pMON81857 151 68 pMON72472aldehyde oxidase Increased yield pMON82218 152 69 pMON72472 cornhypothetical Improved growth under cold pMON78227 protein stress 153 70pMON72472 corn hypothetical Improved growth under cold pMON78904 proteinstress 154 71 pMON72472 Arabidopsis cysteine Increased yield pMON78920proteinase inhibitor 155 72 pMON82053 Arabidopsis cysteine Increasedyield pMON92646 proteinase inhibitor 156 73 pMON72472 ArabidopsisImproved growth under cold pMON78922 hypothetical protein stress 157 74pMON72472 yeast SNF1 - A26030 Improved growth under low pMON78948nitrogen, drought, and/or cold stresses 158 75 pMON72472 soy SNF1-likeprotein 1 Increased yield pMON79660 159 76 pMON72472 soy SNF-likeprotein 2 Enhanced nitrogen use pMON78931 efficiency, enhanced water useefficiency, increased yield 160 77 pMON72472 soy G1760 Increased yieldand pMON82645 enhanced water use efficiency 160 77 Soy G1760 Increasedyield pMON74470 161 78 pMON72472 Rice Glyoxalase II Increased yieldpMON79665 162 79 pMON72472 corn OsPK7-like Enhanced nitrogen usepMON82629 efficiency, enhanced water use efficiency, increased yield 16380 pMON74532 rice phyA with Increased yield pMON81344 Arabidopsis phyCintron I 164 81 pMON82060 rice G975 like1 Improved growth under coldstress 165 82 Corn Phytochrome A Increased yield pMON74916 166 83Arabidopsis G1760 Increased yield pMON73957Selection Methods for Transgenic Plants with Enhanced Agronomic Trait

Within a population of transgenic plants regenerated from plant cellstransformed with the recombinant DNA many plants that survive to fertiletransgenic plants that produce seeds and progeny plants will not exhibitan enhanced agronomic trait. Selection from the population is necessaryto identify one or more transgenic plant cells that can provide plantswith the enhanced trait. Transgenic plants having enhanced traits areselected from populations of plants regenerated or derived from plantcells transformed as described herein by evaluating the plants in avariety of assays to detect an enhanced trait, e.g. enhanced water useefficiency, enhanced cold tolerance, increased yield, enhanced nitrogenuse efficiency, enhanced seed protein and enhanced seed oil. Theseassays also may take many forms including, but not limited to, directscreening for the trait in a greenhouse or field trial or by screeningfor a surrogate trait. Such analyses can be directed to detectingchanges in the chemical composition, biomass, physiological properties,morphology of the plant. Changes in chemical compositions such asnutritional composition of grain can be detected by analysis of the seedcomposition and content of protein, free amino acids, oil, free fattyacids, starch or tocopherols. Changes in biomass characteristics can bemade on greenhouse or field grown plants and can include plant height,stem diameter, root and shoot dry weights; and, for corn plants, earlength and diameter. Changes in physiological properties can beidentified by evaluating responses to stress conditions, for exampleassays using imposed stress conditions such as water deficit, nitrogendeficiency, cold growing conditions, pathogen or insect attack or lightdeficiency, or increased plant density. Changes in morphology can bemeasured by visual observation of tendency of a transformed plant withan enhanced agronomic trait to also appear to be a normal plant ascompared to changes toward bushy, taller, thicker, narrower leaves,striped leaves, knotted trait, chlorosis, albino, anthocyaninproduction, or altered tassels, ears or roots. Other selectionproperties include days to pollen shed, days to silking, leaf extensionrate, chlorophyll content, leaf temperature, stand, seedling vigor,internode length, plant height, leaf number, leaf area, tillering, braceroots, stay green, stalk lodging, root lodging, plant health,barreness/prolificacy, green snap, and pest resistance. In addition,phenotypic characteristics of harvested grain may be evaluated,including number of kernels per row on the ear, number of rows ofkernels on the ear, kernel abortion, kernel weight, kernel size, kerneldensity and physical grain quality. Although the plant cells and methodsof this invention can be applied to any plant cell, plant, seed orpollen, e.g. any fruit, vegetable, grass, tree or ornamental plant, thevarious aspects of the invention are preferably applied to corn,soybean, cotton, canola, alfalfa, wheat and rice plants. In many casesthe invention is applied to corn plants that are inherently resistant todisease from the Mal de Rio Cuarto virus or the Puccina sorghi fungus orboth.

The following examples are included to demonstrate aspects of theinvention, those of skill in the art should, in light of the presentdisclosure, appreciate that many changes can be made in the specificaspects which are disclosed and still obtain a like or similar resultswithout departing from the spirit and scope of the invention.

Example 1 Plant Expression Constructs A. Plant Expression Constructs forCorn Transformation

This example illustrates the construction of plasmids for transferringrecombinant DNA into plant cells which can be regenerated intotransgenic plants of this invention. Primers for PCR amplification ofprotein coding nucleotides of recombinant DNA were designed at or nearthe start and stop codons of the coding sequence, in order to eliminatemost of the 5′ and 3′ untranslated regions. Each recombinant DNA codingfor a protein identified in Table 1 was amplified by PCR prior toinsertion into the insertion site of one of the base vectors asreferenced in Table 1.

A base plant transformation vector pMON65154 was fabricated for use inpreparing recombinant DNA for transformation into corn tissue usingGATEWAY™ Destination plant expression vector systems (available fromInvitrogen Life Technologies, Carlsbad, Calif.). With reference to theelements described in Table 3 below and SEQ ID NO:10024, pMON65154comprises a selectable marker expression cassette and a templaterecombinant DNA expression cassette. The marker expression cassettecomprises a CaMV 35S promoter operably linked to a gene encodingneomycin phosphotransferase II (nptII) followed by a 3′ region of anAgrobacterium tumefaciens nopaline synthase gene (nos). The templaterecombinant DNA expression cassette is positioned tail to tail with themarker expression cassette. The template recombinant DNA expressioncassette comprises 5′ regulatory DNA including a rice actin 1 promoter,exon and intron, followed by a GATEWAY™ insertion site for recombinantDNA, followed by a 3′ region of a potato proteinase inhibitor II (pinII)gene. Once recombinant DNA has been inserted into the insertion site,the plasmid is useful for plant transformation, for example bymicroprojectile bombardment.

TABLE 3 FUNCTION ELEMENT REFERENCE Plant gene of Rice actin 1 promoterU.S. Pat. No. interest expression 5,641,876 cassette Rice actin 1 exon1, U.S. Pat. No. intron 1 enhancer 5,641,876 Gene of interest AttR1GATEWAY ™ insertion site Cloning Technology Instruction Manual CmR geneGATEWAY ™ Cloning Technology Instruction Manual ccdA, ccdB genesGATEWAY ™ Cloning Technology Instruction Manual attR2 GATEWAY ™ CloningTechnology Instruction Manual Plant gene of Potato pinII 3′ region An etal. (1989) Plant interest expression Cell 1: 115-122 cassette Plantselectable CaMV 35S promoter U.S. Pat. No. marker expression 5,858,742cassette nptII selectable marker U.S. Pat. No. 5,858,742 nos 3′ regionU.S. Pat. No. 5,858,742 Maintenance in ColE1 origin of replication E.coli F1 origin of replication Bla ampicillin resistance

A similar base vector plasmid pMON72472 (SEQ ID NO: 10025) wasconstructed for use in Agrobacterium-mediated methods of planttransformation similar to pMON65154 except (a) the 5′ regulatory DNA inthe template recombinant DNA expression cassette was a rice actinpromoter and a rice actin intron, (b) left and right T-DNA bordersequences from Agrobacterium are added with the right border sequence islocated 5′ to the rice actin 1 promoter and the left border sequence islocated 3′ to the 35S promoter and (c) DNA is added to facilitatereplication of the plasmid in both E. coli and Agrobacteriumtumefaciens. The DNA added to the plasmid outside of the T-DNA bordersequences includes an oriV wide host range origin of DNA replicationfunctional in Agrobacterium, a pBR322 origin of replication functionalin E. coli, and a spectinomycin/streptomycin resistance gene forselection in both E. coli and Agrobacterium.

Another base vector pMON82060 (SEQ ID NO: 10026), illustrated in Table4, was assembled using the technology known in the art.

TABLE 4 Coordinates of SEQ ID function name Annotation NO: 10026 AgroB-AGRtu.right border Agro right border sequence, essential for 5235-5591transformation transfer of T-DNA. Gene of P-Os.Act1 Promoter from therice actin gene act1. 5609-7009 interest plant L-Os.Act1 Leader (firstexon) from the rice actin 1 expression gene. cassette I-Os.Act1 Firstintron and flanking UTR exon sequences from the rice actin 1 geneT-St.Pis4 The 3′ non-translated region of the 7084-8026 potatoproteinase inhibitor II gene which functions to direct polyadenylationof the mRNA Plant P-CaMV.35S CaMV 35S promoter 8075-8398 selectableL-CaMV.35S 5′ UTR from the 35S RNA of CaMV marker CR-Ec.nptII-Tn5 nptIIselectable marker that confers 8432-9226 expression resistance toneomycin and kanamycin cassette T-AGRtu.nos A 3′ non-translated regionof the 9255-9507 nopaline synthase gene of Agrobacterium tumefaciens Tiplasmid which functions to direct polyadenylation of the mRNA. AgroB-AGRtu.left border Agro left border sequence, essential for  39-480transformation transfer of T-DNA.. Maintenance OR-Ec.oriV-RK2 Thevegetative origin of replication from 567-963 in E. coli plasmid RK2.CR-Ec.rop Coding region for represser of primer 2472-2663 from the ColE1plasmid. Expression of this gene product interferes with primer bindingat the origin of replication, keeping plasmid copy number low.OR-Ec.ori-ColE1 The minimal origin of replication from 3091-3679 the E.coli plasmid ColE1. P-Ec.aadA-SPC/STR promoter for Tn7adenylyltransferase 4210-4251 (AAD(3″)) CR-Ec.aadA- Coding region forTn7 4252-5040 SPC/STR adenylyltransferase (AAD(3″)) conferringspectinomycin and streptomycin resistance. T-Ec.aadA-SPC/STR 3′ UTR fromthe Tn7 adenylyltransferase 5041-5098 (AAD(3″)) gene of E. coli.

B. Plant Expression Vector for Soybean Transformation

Plasmids for use in transformation of soybean were also prepared.Elements of an exemplary common expression vector plasmid pMON74532 (SEQID NO:10027) are shown in Table 5 below.

TABLE 5 Function Element Reference Agro B-ARGtu.right border Depicker,A. et transformation al (1982) Mol Appl Genet 1: 561-573 AntibioticCR-Ec.aadA-SPC/STR resistance Represser of CR-Ec.rop primers from theColE1 plasmid Origin of OR-Ec.oriV-RK2 replication Agro B-ARGtu.leftborder Barker, R. F. et transformation al (1983) Plant Mol Biol 2:335-350 Plant selectable Promoter with intron and McDowell et al. markerexpression 5′UTR of Arabidopsis act 7 (1996) Plant cassette gene(AtAct7) Physiol. 5′ UTR of Arabidopsis act 7 111: 699-711. gene Intronin 5′UTR of AtAct7 Transit peptide region of Klee, H. J. et ArabidopsisEPSPS al (1987) MGG 210: 437-442 Synthetic CP4 coding region with dicotpreferred codon usage A 3′ UTR of the nopaline U.S. Pat No. synthasegene of 5,858,742 Agrobacterium tumefaciens Ti plasmid Plant gene ofPromoter for 35S RNA from U.S. Pat No. interest expression CaMVcontaining a 5,322,938 cassette duplication of the −90 to −350 regionGene of interest insertion site Cotton E6 3′ end GenBank accessionU30508

Another base vector pMON82053 (SEQ ID NO: 10028), illustrated in Table6, was assembled using the technology known in the art.

TABLE 6 Coordinates of SEQ ID Function Name Annotation NO: 10028 AgroB-AGRtu.left border Agro left border 6144-6585 transforamtion sequence,essential for transfer of T-DNA. Plant P-At.Act7 Promoter from the6624-7861 selectable arabidopsis actin 7 gene marker L-At.Act7 5′UTR ofArabidopsis expression Act7 gene cassette I-At.Act7 Intron from theArabidopsis actin7 gene TS-At.ShkG-CTP2 Transit peptide region of7864-8091 Arabidopsis EPSPS CR-AGRtu.aroA- Synthetic CP4 coding8092-9459 CP4.nno_At region with dicot preferred codon usage.T-AGRtu.nos A 3′ non-translated region 9466-9718 of the nopalinesynthase gene of Agrobacterium tumefaciens Ti plasmid which functions todirect polyadenylation of the mRNA. Gene of P-CaMV.35S-enh Promoter for35S RNA  1-613 interest from CaMV containing a expression duplication ofthe −90 to −350 cassette region. T-Gb.E6-3b 3′ untranslated region 688-1002 from the fiber protein E6 gene of sea-island cotton; AgroB-AGRtu.right border Agro right border 1033-1389 transformationsequence, essential for transfer of T-DNA. Maintenance OR-Ec.oriV-RK2The vegetative origin of 5661-6057 in E. coli replication from plasmidRK2. CR-Ec.rop Coding region for 3961-4152 represser of primer from theColE1 plasmid. Expression of this gene product interferes with primerbinding at the origin of replication, keeping plasmid copy number low.OR-Ec.ori-ColE1 The minimal origin of 2945-3533 replication from the E.coli plasmid ColE1. P-Ec.aadA-SPC/STR romoter for Tn7 2373-2414adenylyltransferase (AAD(3″)) CR-Ec.aadA- Coding region for Tn71584-2372 SPC/STR adenylyltransferase (AAD(3″)) conferring spectinomycinand streptomycin resistance. T-Ec.aadA-SPC/STR 3′ UTR from the Tn71526-1583 adenylyltransferase (AAD(3″))gene of E. coli.

Protein coding segments of recombinant DNA are amplified by PCR prior toinsertion into vectors at the insertion site. Primers for PCRamplification are designed at or near the start and stop codons of thecoding sequence, in order to eliminate most of the 5′ and 3′untranslated regions.

Example 2 Corn Transformation

This example illustrates plant cell transformation methods useful inproducing transgenic corn plant cells, plants, seeds and pollen of thisinvention and the production and identification of transgenic cornplants and seed with an enhanced trait, i.e. enhanced water useefficiency, enhanced cold tolerance, increased yield, enhanced nitrogenuse efficiency, enhanced seed protein and enhanced seed oil. Plasmidvectors were prepared by cloning DNA identified in Table 1 in theidentified base vectors for use in corn transformation of corn plantcells to produce transgenic corn plants and progeny plants, seed andpollen.

For Agrobacterium-mediated transformation of corn embryo cells cornplants of a readily transformable line (designated LH59) is grown in thegreenhouse and ears harvested when the embryos are 1.5 to 2.0 mm inlength. Ears are surface sterilized by spraying or soaking the ears in80% ethanol, followed by air drying. Immature embryos are isolated fromindividual kernels on surface sterilized ears. Prior to inoculation ofmaize cells, Agrobacterium cells are grown overnight at roomtemperature. Immature maize embryo cells are inoculated withAgrobacterium shortly after excision, and incubated at room temperaturewith Agrobacterium for 5-20 minutes. Immature embryo plant cells arethen co-cultured with Agrobacterium for 1 to 3 days at 23° C. in thedark. Co-cultured embryos are transferred to selection media andcultured for approximately two weeks to allow embryogenic callus todevelop. Embryogenic callus is transferred to culture medium containing100 mg/L paromomycin and subcultured at about two week intervals.Transformed plant cells are recovered 6 to 8 weeks after initiation ofselection.

For Agrobacterium-mediated transformation of maize callus immatureembryos are cultured for approximately 8-21 days after excision to allowcallus to develop. Callus is then incubated for about 30 minutes at roomtemperature with the Agrobacterium suspension, followed by removal ofthe liquid by aspiration. The callus and Agrobacterium are co-culturedwithout selection for 3-6 days followed by selection on paromomycin forapproximately 6 weeks, with biweekly transfers to fresh media, andparomomycin resistant callus identified as containing the recombinantDNA in an expression cassette.

For transformation by microprojectile bombardment immature maize embryosare isolated and cultured 3-4 days prior to bombardment. Prior tomicroprojectile bombardment, a suspension of gold particles is preparedonto which the desired recombinant DNA expression cassettes areprecipitated. DNA is introduced into maize cells as described in U.S.Pat. Nos. 5,550,318 and 6,399,861 using the electric discharge particleacceleration gene delivery device. Following microprojectilebombardment, tissue is cultured in the dark at 27 degrees C. Additionaltransformation methods and materials for making transgenic plants ofthis invention, for example, various media and recipient target cells,transformation of immature embryos and subsequence regeneration offertile transgenic plants are disclosed in U.S. Pat. Nos. 6,194,636 and6,232,526 and U.S. patent application Ser. No. 09/757,089, which areincorporated herein by reference.

To regenerate transgenic corn plants a callus of transgenic plant cellsresulting from transformation is placed on media to initiate shootdevelopment in plantlets which are transferred to potting soil forinitial growth in a growth chamber at 26 degrees C. followed by a mistbench before transplanting to 5 inch pots where plants are grown tomaturity. The regenerated plants are self fertilized and seed isharvested for use in one or more methods to select seed, seedlings orprogeny second generation transgenic plants (R2 plants) or hybrids, e.g.by selecting transgenic plants exhibiting an enhanced trait as comparedto a control plant.

Transgenic corn plant cells were transformed with recombinant DNA fromeach of the genes identified in Table 1. Progeny transgenic plants andseed of the transformed plant cells were screened for enhanced water useefficiency, enhanced cold tolerance, increased yield, enhanced nitrogenuse efficiency, enhanced seed protein and enhanced seed oil as reportedin Example 5.

Example 3 Soybean Transformation

This example illustrates plant transformation useful in producing thetransgenic soybean plants of this invention and the production andidentification of transgenic seed for transgenic soybean having enhancedwater use efficiency, enhanced cold tolerance, increased yield, enhancednitrogen use efficiency, enhanced seed protein and enhanced seed oil.

For Agrobacterium mediated transformation, soybean seeds are germinatedovernight and the meristem explants excised. The meristems and theexplants are placed in a wounding vessel. Soybean explants and inducedAgrobacterium cells from a strain containing plasmid DNA with the geneof interest cassette and a plant selectable marker cassette are mixed nolater than 14 hours from the time of initiation of seed germination andwounded using sonication. Following wounding, explants are placed inco-culture for 2-5 days at which point they are transferred to selectionmedia for 6-8 weeks to allow selection and growth of transgenic shoots.Trait positive shoots are harvested approximately 6-8 weeks and placedinto selective rooting media for 2-3 weeks. Shoots producing roots aretransferred to the greenhouse and potted in soil. Shoots that remainhealthy on selection, but do not produce roots are transferred tonon-selective rooting media for an additional two weeks. Roots from anyshoots that produce roots off selection are tested for expression of theplant selectable marker before they are transferred to the greenhouseand potted in soil. Additionally, a DNA construct can be transferredinto the genome of a soybean cell by particle bombardment and the cellregenerated into a fertile soybean plant as described in U.S. Pat. No.5,015,580, herein incorporated by reference.

Transgenic soybean plant cells were transformed with recombinant DNAfrom each of the genes identified in Table 1. Progeny transgenic plantsand seed of the transformed plant cells were screened for enhanced wateruse efficiency, enhanced cold tolerance, increased yield, enhancednitrogen use efficiency, enhanced seed protein and enhanced seed oil asreported in Example 5.

Example 4 Homolog Identification

This example illustrates the identification of homologs of proteinsencoded by the DNA identified in Table 1 which is used to providetransgenic seed and plants having enhanced agronomic traits. From thesequence of the homologs, homologous DNA sequence can be identified forpreparing additional transgenic seeds and plants of this invention withenhanced agronomic traits.

An “All Protein Database” was constructed of known protein sequencesusing a proprietary sequence database and the National Center forBiotechnology Information (NCBI) non-redundant amino acid database(nr.aa). For each organism from which a polynucleotide sequence providedherein was obtained, an “Organism Protein Database” was constructed ofknown protein sequences of the organism; it is a subset of the AllProtein Database based on the NCBI taxonomy ID for the organism.

The All Protein Database was queried using amino acid sequences providedherein as SEQ ID NO:84 through SEQ ID NO:166 using NCBI “blastp” programwith E-value cutoff of 1e-8. Up to 1000 top hits were kept, andseparated by organism names. For each organism other than that of thequery sequence, a list was kept for hits from the query organism itselfwith a more significant E-value than the best hit of the organism. Thelist contains likely duplicated genes of the polynucleotides providedherein, and is referred to as the Core List. Another list was kept forall the hits from each organism, sorted by E-value, and referred to asthe Hit List.

The Organism Protein Database was queried using polypeptide sequencesprovided herein as SEQ ID NO:84 through SEQ ID NO:166 using NCBI“blastp” program with E-value cutoff of 1e-4. Up to 1000 top hits werekept. A BLAST searchable database was constructed based on these hits,and is referred to as “SubDB”. SubDB was queried with each sequence inthe Hit List using NCBI “blastp” program with E-value cutoff of 1e-8.The hit with the best E-value was compared with the Core List from thecorresponding organism. The hit is deemed a likely ortholog if itbelongs to the Core List, otherwise it is deemed not a likely orthologand there is no further search of sequences in the Hit List for the sameorganism. Homologs from a large number of distinct organisms wereidentified and are reported by amino acid sequences of SEQ ID NO: 167through SEQ ID NO: 10023. These relationship of proteins of SEQ ID NO:84through 166 and homologs of SEQ ID NO:167 through 10023 is identified inTable 2. The source organism for each homolog is found in the SequenceListing.

TABLE 2 PEP SEQ ID NO: homolog SEQ ID NOs 84: 4274 4007 7537 1472 24651788 1873 8538 2486 2101 2090 3705 513 7264 6280 4902 2624 8820 16145907 8247 2717 4147 5559 1631 7278 6566 6687 2116 9018 192 2002 5150 3226314 6458 6281 1285 7292 4226 4543 2496 9903 1478 554 5383 7751 24844954 7695 5821 6271 3339 443 8542 1561 2321 5876 6877 3452 2879 34972097 4257 7449 7281 3708 4513 2001 4425 9319 4133 6686 2146 9698 10362026 1292 5566 181 6951 9794 2439 2621 5202 878 8081 1392 1950 9999 43922121 7824 2367 5102 6717 1541 9444 7051 529 4096 602 8266 85: 1163 39549565 5913 8096 1310 3871 3019 2926 1456 2770 4461 2570 5099 7946 37009665 1600 7270 7312 6531 9978 8803 8920 4917 6067 6352 6902 2025 25164213 9446 8483 5404 2213 4311 3724 9926 9599 3835 727 8396 190 3701 7478706 4038 7149 5413 1538 8094 9467 7385 7520 7275 3299 3658 86: 2511 25137067 7055 5647 9608 9399 4420 9867 4564 2527 7769 2323 347 6509 20525258 4504 5363 3847 329 7133 1751 3243 8135 4767 5558 2719 6177 61616180 1606 3066 514 7725 4747 2868 3953 3995 9218 8245 1471 1050 46029788 5705 1043 87: 7338 2565 1372 619 8819 7803 7216 9263 8478 7286 20518010 4629 2569 8521 7659 6081 6080 2727 1944 5731 7616 8198 8166 63129586 2010 7801 4694 4265 3928 9925 1675 6099 5725 1040 5933 270 41356356 8593 7015 3351 9045 5105 9655 3874 5951 2184 7921 9476 3408 70951214 9077 3211 7050 7106 4788 3534 3093 7715 88: 9004 8450 3918 3721 5168506 8664 3458 6365 2464 1564 4322 7760 3673 7547 2603 8146 1755 79194542 436 2278 4913 2453 9651 2319 3659 678 4640 3600 4171 1156 1807 57656619 2992 354 8233 2386 9454 9453 8837 1238 6971 7874 6538 8258 13711609 3120 3437 8825 7158 5623 1313 7335 6137 3691 8239 415 7580 51478818 6282 4612 543 6639 9686 7662 7683 7682 7664 5278 5260 8016 25582566 2530 9515 5921 8962 3892 7174 6793 6936 6938 8284 5225 9323 29324932 5328 6697 6602 5109 9625 1876 7435 7758 1719 662 6913 4095 55634919 8188 6804 360 9790 7742 7745 2584 8776 8004 862 6690 8757 5193 66189595 225 4815 5192 1055 4061 4017 9781 9955 8231 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6262 2659 820 6646 9005 4294 6457 6451 2869 2767 77461732 1738 1734 1740 1617 6714 8105 7317 7318 1709 1731 6558 7716 79717968 1210 1248 925 6580 1247 1273 1274 3369 2586 3664 91: 7402 7398 35833592 4729 6500 6496 2752 215 7280 3139 3136 7439 7440 7424 9236 92373124 3128 3938 177 1956 1982 1979 7291 7290 4868 6436 7355 7315 72977279 794 7341 7272 7262 290 304 289 291 288 7324 7321 3947 6330 66616658 4296 3753 8555 1943 8457 3157 7565 7568 7088 5977 4467 3998 31853181 3152 3155 7534 7516 7244 1847 7442 7468 6000 8494 375 1057 97736372 7369 957 2685 3508 3586 3087 8866 5749 1377 2317 723 4586 2111 47034413 3687 8541 9052 1793 6037 2164 2626 6824 5207 3238 7618 9974 99731815 1813 206 4540 2230 6716 5597 2206 2232 2200 5599 1187 3435 96729040 9039 2881 7223 2377 2292 7024 6395 4348 4880 744 8064 5301 84928447 4312 4317 8472 1863 7956 6694 8324 7358 381 5710 3028 2182 70203016 531 5699 9003 7798 7421 2299 787 1905 3641 1934 1893 1889 7496 50367100 3584 3055 2018 2043 2042 7345 1084 1086 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67011803 4281 816 2806 396 2594 3089 97: 6736 292 4996 5983 8898 6886 88451312 9032 1586 9964 9971 6476 455 357 6653 3756 4574 3311 920 3295 34439601 6906 5214 5215 5216 5217 7960 7945 7009 8847 4936 6359 5296 65712233 3288 8736 5420 4531 9662 4891 7545 7948 7980 2201 9853 8153 48618591 1746 7233 8693 3252 8690 709 5069 9505 98: 9658 9420 3779 6609 30062471 3567 4983 434 3565 10008 8348 5728 8033 8302 5426 8854 6705 41604243 9780 9744 8244 4469 317 152 522 6171 3571 3894 8512 2442 9642 62553980 3725 99: 9060 9635 3511 3575 1890 1852 7041 4217 2648 156 2833 84567232 8503 5709 8742 6107 9824 9121 1142 886 9105 8210 2910 100: 95261911 5622 9279 2726 1935 4608 7672 9536 4429 260 4235 356 5588 4995 24265771 4203 1942 6086 3697 2322 9359 1534 9550 1034 8012 7732 7017 1448101: 4840 6812 777 7939 1059 6995 5721 4881 5741 9047 566 3050 8123 25908060 4040 6354 2078 2081 3304 2079 3305 2734 1841 9901 9621 1060 63853676 2300 1773 9674 4263 7267 9920 7962 1042 6416 8813 2382 4599 56703931 4207 2985 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7903 43724385 7928 7959 7976 7951 7981 7984 8019 8006 8025 8020 4368 4366 43434363 4382 7894 8026 4334 4332 4822 7117 8003 7923 7983 4336 3180 31583129 2245 3086 3137 2290 3183 3186 3188 8700 8701 2393 2368 3117 47394972 4763 4766 4772 4806 2241 2289 2305 2270 2264 2267 2284 2212 22376357 4975 4756 9294 8327 5259 5256 5867 4794 8493 8497 3144 3142 11137676 7658 7675 3548 3551 4329 8490 9196 9198 4848 9152 9155 9145 91449135 9136 9204 8385 2226 2228 2231 2234 3552 3553 7721 9640 2515 2518277 3910 3018 6615 3355 6975 6980 6976 7713 7000 3517 4790 4802 48303405 2451 4011 4833 5828 4712 4805 4771 4746 5318 4713 4793 4835 53195317 4742 4764 1421 1424 9606 6625 1969 1971 1434 8672 8669 3130 20132029 9516 8726 8730 9336 9339 9337 6631 4829 6934 1743 5745 1958 78696426 1986 1987 7340 5037 4785 4789 912 3876 3873 3891 3102 8807 86268642 3132 4854 9232 9233 9202 9132 1418 1419 9199 9200 9184 1628 11721175 1177 9360 4765 9855 163 5084 6821 5061 671 657 669 655 5314 50678113 8117 298 1989 1991 4796 4808 6244 6488 6490 6503 4743 3312 68224067 5279 9156 9158 5205 3147 3168 4856 3156 1548 1798 1546 208 89448933 8949 8276 8279 8281 8203 6005 8253 8992 8995 8996 8972 8991 95198197 5917 5978 8223 5984 5936 8316 5956 5958 8226 8229 8300 9098 90999560 9563 9580 9579 9582 9583 8919 7418 7794 1587 1579 7692 5273 15935261 4710 4279 1547 3895 3893 3907 3081 4195 5210 4758 4197 4727 88328916 8851 8857 8894 8892 8871 8865 8849 8872 8889 8874 8927 8932 89308895 8909 4770 1062 7681 7691 5213 5226 5157 5160 1375 1400 1458 14621482 2037 2041 9172 5321 1396 1415 4714 4741 4966 4970 5000 5002 47455003 4760 4762 5005 4769 4798 5064 7114 1379 1352 1341 1453 3929 39253080 8750 7430 9401 9403 2007 2059 2062 2064 4857 8675 8677 4940 49654973 5004 5039 5041 5045 705 707 691 693 692 2033 2035 4740 4709 47494748 4803 5312 5313 5315 1745 8270 166: 7398 6484 6449 4729 6500 64966480 6477 4169 7256 7258 2351 6605 6606 7280 9607 7439 9571 9415 67119236 9237 6585 177 1949 7291 7290 4868 6438 6265 7293 7295 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Example 5 Selection of Transgenic Plants with Enhanced AgronomicTrait(s)

This example illustrates identification of plant cells of the inventionby screening derived plants and seeds for enhanced trait. Transgeniccorn seed and plants with recombinant DNA identified in Table 1 wereprepared by plant cells transformed with DNA that was stably integratedinto the genome of the corn cell. The transgenic seed, plantlets andprogeny plants were selected using the methods that measure Transgeniccorn plant cells were transformed with recombinant DNA from each of thegenes identified in Table 1. Progeny transgenic plants and seed of thetransformed plant cells were screened for enhanced water use efficiency,enhanced cold tolerance, increased yield, enhanced nitrogen useefficiency, enhanced seed protein and enhanced seed oil as compared tocontrol plants.

A. Selection for enhanced Nitrogen Use Efficiency

The physiological efficacy of transgenic corn plants (tested as hybrids)can be tested for nitrogen use efficiency (NUE) traits in ahigh-throughput nitrogen (N) selection method. The collected data arecompared to the measurements from wildtype controls using a statisticalmodel to determine if the changes are due to the transgene. Raw datawere analyzed by SAS software. Results shown herein are the comparisonof transgenic plants relative to the wildtype controls.

(1) Media Preparation for Planting a NUE Protocol

Planting materials used: Metro Mix 200 (vendor: Hummert) Cat. #10-0325,Scotts Micro Max Nutrients (vendor: Hummert) Cat. #07-6330, OS 4⅓″×3⅞″pots (vendor: Hummert) Cat. #16-1415, OS trays (vendor: Hummert) Cat.#16-1515, Hoagland's macronutrients solution, Plastic 5″ stakes (vendor:Hummert) yellow Cat. #49-1569, white Cat. #49-1505, Labels with numbersindicating material contained in pots. Fill 500 pots to rim with MetroMix 200 to a weight of ˜140 g/pot. Pots are filled uniformly by using abalancer. Add 0.4 g of Micro Max nutrients to each pot. Stir ingredientswith spatula to a depth of 3 inches while preventing material loss.

(2) Planting a NUE Selection in the Greenhouse

(a) Seed Germination—Each pot is lightly atered twice using reverseosmosis purified water. The first watering is scheduled to occur justbefore planting; and the second watering, after the seed has beenplanted in the pot. Ten Seeds of each entry (1 seed per pot) are plantedto select eight healthy uniform seedlings. Additional wild type controlsare planted for use as border rows. Alternatively, 15 seeds of eachentry (1 seed per pot) are planted to select 12 healthy uniformseedlings (this larger number of plantings is used for the second, orconfirmation, planting). Place pots on each of the 12 shelves in theConviron growth chamber for seven days. This is done to allow moreuniform germination and early seedling growth. The following growthchamber settings are 25° C./day and 22° C./night, 14 hours light and tenhours dark, humidity ˜80%, and light intensity ˜350 μmol/m²/s (at potlevel). Watering is done via capillary matting similar to greenhousebenches with duration of ten minutes three times a day.

(b) Seedling transfer—After seven days, the best eight or 12 seedlingsfor the first or confirmation pass runs, respectively, are chosen andtransferred to greenhouse benches. The pots are spaced eight inchesapart (center to center) and are positioned on the benches using thespacing patterns printed on the capillary matting. The Vattex mattingcreates a 384-position grid, randomizing all range, row combinations.Additional pots of controls are placed along the outside of theexperimental block to reduce border effects.

Plants are allowed to grow for 28 days under the low N run or for 23days under the high N run. The macronutrients are dispensed in the formof a macronutrient solution (see composition below) containing preciseamounts of N added (2 mM NH₄NO₃ for limiting N selection and 20 mMNH₄NO₃ for high N selection runs). Each pot is manually dispensed 100 mlof nutrient solution three times a week on alternate days starting ateight and ten days after planting for high N and low N runs,respectively. On the day of nutrient application, two 20 min wateringsat 05:00 and 13:00 are skipped. The vattex matting should be changedevery third run to avoid N accumulation and buildup of root matter.Table 7 shows the amount of nutrients in the nutrient solution foreither the low or high nitrogen selection.

TABLE 7 2 mM NH₄NO₃ 20 mM NH₄NO₃ (Low Nitrogen (high Nitrogen GrowthCondition, Growth Condition, Low N) High N) Nutrient Stock mL/L mL/L 1MNH₄NO₃ 2 20 1M KH₂PO₄ 0.5 0.5 1M MgSO₄•7H₂O 2 2 1M CaCl₂ 2.5 2.5 1MK₂SO₄ 1 1 Note: Adjust pH to 5.6 with HCl or KOH

(c) Harvest Measurements and Data Collection—After 28 days of plantgrowth for low N runs and 23 days of plant growth for high N runs, thefollowing measurements are taken (phenocodes in parentheses): totalshoot fresh mass (g) (SFM) measured by Sartorius electronic balance, V6leaf chlorophyll measured by Minolta SPAD meter (relative units) (LC),V6 leaf area (cm²) (LA) measured by a Li-Cor leaf area meter, V6 leaffresh mass (g) (LFM) measured by Sartorius electronic balance, and V6leaf dry mass (g) (LDM) measured by Sartorius electronic balance. Rawdata were analyzed by SAS software. Results shown are the comparison oftransgenic plants relative to the wildtype controls.

To take a leaf reading, samples were excised from the V6 leaf. Sincechlorophyll meter readings of corn leaves are affected by the part ofthe leaf and the position of the leaf on the plant that is sampled, SPADmeter readings were done on leaf six of the plants. Three measurementsper leaf were taken, of which the first reading was taken from a pointone-half the distance between the leaf tip and the collar and halfwayfrom the leaf margin to the midrib while two were taken toward the leaftip. The measurements were restricted in the area from ½ to ¾ of thetotal length of the leaf (from the base) with approximately equalspacing between them. The average of the three measurements was takenfrom the SPAD machine.

Leaf fresh mass is recorded for an excised V6 leaf, the leaf is placedinto a paper bag. The paper bags containing the leaves are then placedinto a forced air oven at 80° C. for 3 days. After 3 days, the paperbags are removed from the oven and the leaf dry mass measurements aretaken.

From the collected data, two derived measurements are made: (1) Leafchlorophyll area (LCA), which is a product of V6 relative chlorophyllcontent and its leaf area (relative units). Leaf chlorophyll area=leafchlorophyll X leaf area. This parameter gives an indication of thespread of chlorophyll over the entire leaf area; (2) specific leaf area(LSA) is calculated as the ratio of V6 leaf area to its dry mass (cm²/gdry mass), a parameter also recognized as a measure of NUE. The data areshown in Table 8.

TABLE 8 Leaf chlorophyll area Leaf chlorophyll Shoot fresh mass MeanMean Mean PEP of of of SEQ Construct Event Percent con- Percent con-Percent con- ID ID ID change Mean trols P-value change Mean trolsP-value change Mean trols P-value 91 PMON73816 ZM_M37183 4 3688.433558.85 0.221 3 24.54 23.73 0.0722 5 48.04 45.92 0.1289 PMON73816ZM_M37183 15 5963.14 5180.33 0 12 31.72 28.41 0 16 48.24 41.48 1.00E−04PMON73816 ZM_M37183 8 4796 4439.2 0.0438 3 27.1 26.2 0.2569 23 55.2 44.80 PMON73816 ZM_M37188 12 4002.73 3558.85 0 13 26.86 23.73 0 4 47.8345.92 0.1707 PMON73816 ZM_M37188 13 5832.79 5180.33 3.00E−04 12 31.7328.41 0 11 46.25 41.48 0.0046 PMON73816 ZM_M37188 −9 4037.7 4439.20.0234 −1 26 26.2 0.7492 −10 40.4 44.8 0.0144 PMON73816 ZM_M37197 45375.2 5180.33 0.2694 1 28.81 28.41 0.5194 17 48.42 41.48 0 PMON73816ZM_M37197 21 5374.8 4439.2 0 14 29.9 26.2 0 30 58.4 44.8 0 PMON73816ZM_M37197 5 3733.33 3558.85 0.0996 1 24.02 23.73 0.522 5 48.42 45.920.0742 100 PMON75511 ZM_M44958 18.1 5065.43 4287.52 1.00E−04 13.9 29.4425.86 0 12 44.22 39.48 0.0096 PMON75511 ZM_M44958 7.3 8006.21 7460.910.0071 5.5 40.63 38.5 0.0072 0 67.53 67.56 0.9892 PMON75511 ZM_M449618.2 4639.06 4287.52 0.0583 5.8 27.36 25.86 0.0449 6.7 42.13 39.48 0.1258PMON75511 ZM_M44961 4.7 7810.27 7460.91 0.0947 4.9 40.41 38.5 0.0195 4.970.87 67.56 0.1511 PMON75511 ZM_M46591 5.1 4504.72 4287.52 0.2951 5.527.27 25.86 0.0734 −4.5 37.69 39.48 0.3276 PMON75511 ZM_M46591 −4.37142.88 7460.91 0.1149 −1.4 37.98 38.5 0.4997 8 72.98 67.56 0.0151PMON75511 ZM_M46601 12.3 4813.03 4287.52 0.0117 4.7 27.07 25.86 0.149422.4 48.31 39.48 0 PMON75511 ZM_M46601 7.7 8036.73 7460.91 0.0045 540.44 38.5 0.014 0.3 67.76 67.56 0.93 114 PMON75980 ZM_M53387 −8 3998.294368.22 0.0065 2 24.35 23.8 0.3237 −18 37.79 45.85 0 PMON75980 ZM_M53389−10 3323.6 3691.69 0.0189 −3 23.05 23.65 0.3551 −8 30.6 33.21 0.0804PMON75980 ZM_M53389 −5 4139.75 4368.22 0.1038 −2 23.42 23.8 0.4834 −1041.22 45.85 0.0031 PMON75980 ZM_M53390 8 4728.73 4368.22 0.0188 5 25.0723.8 0.0272 −3 44.65 45.85 0.4407 PMON75980 ZM_M53390 10 4044.06 3691.690.0245 2 24.24 23.65 0.3703 9 36.29 33.21 0.0398 PMON75980 ZM_M53392 274679.18 3691.69 0 10 26.06 23.65 3.00E−04 27 42.31 33.21 0 PMON75980ZM_M53392 2 4446.67 4368.22 0.5757 4 24.88 23.8 0.0534 3 47.36 45.850.3298 PMON75980 ZM_M53396 13 4948.67 4368.22 0 7 25.37 23.8 0.0068 849.32 45.85 0.0259 PMON75980 ZM_M53396 16 4271.59 3691.69 2.00E−04 424.7 23.65 0.109 13 37.46 33.21 0.0046 PMON75980 ZM_M53397 1 4411.54368.22 0.7574 1 24.06 23.8 0.6707 −6 43.08 45.85 0.0992 PMON75980ZM_M53398 2 4476.43 4368.22 0.4235 7 25.36 23.8 0.0052 −6 43.12 45.850.0792 103 PMON78949 ZM_M63936 −2.1 4587.66 4686.12 0.4835 3.3 30.3529.37 0.1605 −6.1 32.65 34.77 0.0457 PMON78949 ZM_M63936 −2.1 3863.183946.32 0.4391 −0.6 28.37 28.55 0.7352 8.7 45.14 41.55 0.0077 PMON78949ZM_M63941 7.5 5037.73 4686.12 0.0128 3.9 30.51 29.37 0.1021 7.4 37.3334.77 0.0158 PMON78949 ZM_M63941 −1.9 3871.03 3946.32 0.4835 −2.5 27.8328.55 0.1742 9.8 45.63 41.55 0.0036 PMON78949 ZM_M63942 7.5 5036.214686.12 0.0132 6.4 31.26 29.37 0.007 9.2 37.98 34.77 0.0025 PMON78949ZM_M63942 13 4459.25 3946.32 0 7.6 30.73 28.55 0 9.2 45.37 41.55 0.0047PMON78949 ZM_M63944 4.3 4887.29 4686.12 0.1528 4.9 30.81 29.37 0.0393−6.6 32.48 34.77 0.0306 PMON78949 ZM_M63944 0.8 3979.53 3946.32 0.75710.4 28.66 28.55 0.8318 −0.9 41.17 41.55 0.7776 108 PMON79709 ZM_M51983 35110.49 4947.82 0.1855 6 28.18 26.59 0.0012 4 46.1 44.36 0.076 PMON79709ZM_M51983 2 6011.13 5906.6 0.6174 3 28.75 27.9 0.2078 16 62.26 53.532.00E−04 PMON79709 ZM_M51983 0.9 5829.16 5776.02 0.7681 −0.7 30.24 30.450.7671 −3.1 45.46 46.92 0.4097 PMON79709 ZM_M51985 0 5773.16 5776.020.988 −0.2 30.38 30.45 0.9183 −1.6 46.16 46.92 0.682 PMON79709 ZM_M519857 6301.05 5906.6 0.0602 3 28.81 27.9 0.1763 16 62.11 53.53 2.00E−04PMON79709 ZM_M51985 6 5263.87 4947.82 0.0079 6 28.07 26.59 0.0026 345.48 44.36 0.2555 PMON79709 ZM_M52025 3 5075.34 4947.82 0.2817 4 27.5826.59 0.0415 4 46.33 44.36 0.052 PMON79709 ZM_M52025 3.2 5959.63 5776.020.3087 −1.7 29.93 30.45 0.4617 1 47.38 46.92 0.7983 PMON79709 ZM_M5202521 7124.16 5906.6 0 14 31.74 27.9 0 20 64.48 53.53 0 PMON79709 ZM_M527106 6240.85 5906.6 0.1109 10 30.6 27.9 1.00E−04 9 58.5 53.53 0.0321PMON79709 ZM_M52710 8 5339.8 4947.82 0.001 7 28.46 26.59 1.00E−04 345.82 44.36 0.1373 PMON79709 ZM_M52710 3.8 5995.36 5776.02 0.2241 3.631.55 30.45 0.1214 −4.1 45 46.92 0.2779 PMON79709 ZM_M52720 7.4 6201.465776.02 0.0188 5.2 32.04 30.45 0.0258 6.1 49.8 46.92 0.1242 PMON79709ZM_M52720 7 5280.25 4947.82 0.0053 7 28.39 26.59 2.00E−04 −5 42.31 44.360.0357 PMON79709 ZM_M52720 12 6617.79 5906.6 8.00E−04 9 30.28 27.99.00E−04 3 55.01 53.53 0.5222 96 PMON80270 ZM_M55967 5.2 6306.34 5993.370.0376 4.3 30.64 29.39 0.028 7.3 54.7 50.98 0.0017 PMON80270 ZM_M559676.6 5.33 5 0.0666 6.7 33.48 31.38 0.0075 6 44.75 42.21 0.0627 PMON80270ZM_M55968 16.6 5.83 5 0 5.7 33.17 31.38 0.0421 17.5 49.6 42.21 0PMON80270 ZM_M55968 −1 5930.77 5993.37 0.6873 −0.5 29.25 29.39 0.80587.7 54.89 50.98 0.001 PMON80270 ZM_M55969 −4.1 5749.51 5993.37 0.10480.3 29.47 29.39 0.892 4.7 53.36 50.98 0.0427 PMON80270 ZM_M55969 5 5.255 0.1118 4.1 32.66 31.38 0.1464 8 45.58 42.21 0.0139 PMON80270 ZM_M55970−2.3 5855.83 5993.37 0.3595 1.3 29.76 29.39 0.5246 4.4 53.2 50.98 0.0504PMON80270 ZM_M55970 2.6 5.13 5 0.4257 −2.5 30.58 31.38 0.3062 2.9 43.4542.21 0.3616 PMON80270 ZM_M55971 −4 5754.31 5993.37 0.1118 0.7 29.6129.39 0.7 1.8 51.92 50.98 0.4075 PMON80270 ZM_M55971 6 5.3 5 0.0728 4.832.89 31.38 0.0536 6 44.74 42.21 0.064 PMON80270 ZM_M55972 −1 5933.485993.37 0.6897 −0.3 29.29 29.39 0.8631 3.6 52.81 50.98 0.1193 PMON80270ZM_M55972 13.8 5.69 5 0 5.1 32.99 31.38 0.0397 9.4 46.19 42.21 0.0037PMON80270 ZM_M56524 8 5.4 5 0.0364 5.1 32.98 31.38 0.0413 15.5 48.7442.21 0 PMON80270 ZM_M56524 −1.4 5908.18 5993.37 0.5702 1 29.67 29.390.6255 6.3 54.18 50.98 0.0067 PMON80270 ZM_M56526 −2.7 5829.79 5993.370.276 −1.4 28.98 29.39 0.4744 2.5 52.23 50.98 0.2681 PMON80270 ZM_M5652620 6 5 0 0.5 31.54 31.38 0.8352 13.8 48.05 42.21 0 PMON80270 ZM_M565271.2 6063.11 5993.37 0.6421 −0.2 29.32 29.39 0.8978 5.6 53.82 50.980.0126 PMON80270 ZM_M56527 2.2 5.11 5 0.489 2.4 32.14 31.38 0.3294 4.143.95 42.21 0.2012 118 PMON80461 ZM_M52932 24.5 8417.13 6759.85 0 13.434.66 30.57 0 25.7 76.5 60.88 0 PMON80461 ZM_M52932 6 7095.13 6713.170.0553 3 30.63 29.82 0.294 −1 54.05 54.73 0.653 PMON80461 ZM_M52932 14877.13 4816.31 0.5834 2 29.24 28.65 0.2351 −2 30.75 31.34 0.4187PMON80461 ZM_M52932 −4.5 5830.38 6107.25 0.1599 −1.1 29.45 29.77 0.6468−2.7 37.58 38.63 0.5145 PMON80461 ZM_M52932 −9 4808.1 5269.64 0.0084 130.86 30.68 0.7905 2 35.8 35.13 0.4119 PMON80461 ZM_M52932 8.2 5068.244686.12 0.0069 10 32.31 29.37 0 −6 32.68 34.77 0.0483 PMON80461ZM_M52932 14.3 4511.99 3946.32 0 6.5 30.42 28.55 5.00E−04 11 46.12 41.557.00E−04 PMON80461 ZM_M53218 −14.6 5773.62 6759.85 1.00E−04 −5.6 28.8730.57 0.0168 −16.4 50.92 60.88 1.00E−04 PMON80461 ZM_M53218 7 7166.446713.17 0.0231 5 31.33 29.82 0.0501 9 59.48 54.73 0.002 PMON80461ZM_M53218 2 4908.21 4816.31 0.4075 3 29.55 28.65 0.072 3 32.25 31.340.1908 PMON80461 ZM_M53218 −9 4808.4 5269.64 0.0085 −2 30.04 30.680.3563 −2 34.52 35.13 0.4641 PMON80461 ZM_M53218 8.2 5071.81 4686.120.0064 6.2 31.19 29.37 0.0096 0.4 34.91 34.77 0.8893 PMON80461 ZM_M532181.7 6211.2 6107.25 0.6164 −1.5 29.33 29.77 0.5225 0.9 38.97 38.63 0.8332PMON80461 ZM_M53218 1.1 3987.88 3946.32 0.6988 1.3 28.92 28.55 0.484 1.342.07 41.55 0.6981 PMON80461 ZM_M53235 3 4955.98 4816.31 0.2084 1 28.9328.65 0.5828 0 31.45 31.34 0.8709 PMON80461 ZM_M53235 20.2 8122.466759.85 0 13.8 34.79 30.57 0 17.3 71.4 60.88 0 PMON80461 ZM_M53235 36907.56 6713.17 0.3282 5 31.36 29.82 0.0447 1 55.05 54.73 0.8357PMON80461 ZM_M53503 2 4921.37 4816.31 0.3438 8 30.95 28.65 0 3 32.3231.34 0.1605 PMON80461 ZM_M53503 14.9 7763.72 6759.85 1.00E−04 10.433.77 30.57 0 25.9 76.63 60.88 0 PMON80461 ZM_M53503 7 7197.24 6713.170.0154 6 31.54 29.82 0.0255 12 61.48 54.73 0 PMON80461 ZM_M53504 −16666.94 6713.17 0.816 1 29.98 29.82 0.8413 10 60.29 54.73 6.00E−04PMON80461 ZM_M53504 −1 4748.6 4816.31 0.5416 −1 28.4 28.65 0.6231 −230.82 31.34 0.4559 PMON80461 ZM_M53504 −15.3 5724.41 6759.85 0 −8.627.93 30.57 2.00E−04 −21 48.11 60.88 0 PMON80461 ZM_M53848 2 4897.294816.31 0.4654 4 29.87 28.65 0.0153 −2 30.63 31.34 0.3077 PMON80461ZM_M53848 −15.3 5722.73 6759.85 0 −5.6 28.87 30.57 0.0168 −24.1 46.1960.88 0 PMON80461 ZM_M53848 3 6882.64 6713.17 0.394 7 31.86 29.82 0.0082 56 54.73 0.4059 PMON80461 ZM_M54282 0 4800.09 4816.31 0.8878 2 29.3128.65 0.2011 −1 30.98 31.34 0.6261 PMON80461 ZM_M54282 −2 6592.766713.17 0.5446 −2 29.35 29.82 0.5372 3 56.57 54.73 0.2552 PMON80461ZM_M54282 −12.7 5900.82 6759.85 7.00E−04 −4.9 29.07 30.57 0.0346 −19.848.83 60.88 0 PMON80461 ZM_M54284 7 7155.9 6713.17 0.0265 5 31.2 29.820.0723 1 55.01 54.73 0.855 PMON80461 ZM_M54284 19.2 8060.14 6759.85 09.7 33.55 30.57 0 16.4 70.88 60.88 1.00E−04 PMON80461 ZM_M54284 5 5052.84816.31 0.0404 1 28.94 28.65 0.5692 3 32.14 31.34 0.2488 PMON80461ZM_M55266 −2.4 5962.4 6107.25 0.4616 0.2 29.81 29.77 0.9457 −6.7 36.0438.63 0.1098 PMON80461 ZM_M55957 5 6414.71 6107.25 0.1187 2.9 30.6329.77 0.2128 −3.7 37.21 38.63 0.3528 PMON80461 ZM_M56233 2.7 6270.896107.25 0.4056 5 31.25 29.77 0.0426 −0.7 38.38 38.63 0.8653 PMON80461ZM_M56728 3.8 6338.35 6107.25 0.2405 4 30.96 29.77 0.0831 −0.5 38.4338.63 0.8911 102 PMON80542 ZM_M57107 −3.8 5766.93 5993.37 0.1461 −0.229.34 29.39 0.9327 6.1 54.07 50.98 0.0089 PMON80542 ZM_M57107 14.2 5.715 0 1.2 31.75 31.38 0.6312 12.3 47.4 42.21 2.00E−04 PMON80542 ZM_M57119−8 5512.76 5993.37 0.0015 −1.1 29.08 29.39 0.5896 4.6 53.34 50.98 0.0375PMON80542 ZM_M57119 11.6 5.58 5 5.00E−04 5.1 32.96 31.38 0.0429 16.249.03 42.21 0 PMON80542 ZM_M57120 2.6 5.13 5 0.4257 2.5 32.16 31.380.3138 −1.1 41.75 42.21 0.7377 PMON80542 ZM_M57120 −3.1 5807.66 5993.370.2163 0.2 29.46 29.39 0.9036 0.1 51.04 50.98 0.9595 PMON80542 ZM_M57121−2.7 5829.33 5993.37 0.2746 1.9 29.94 29.39 0.3311 8.4 55.24 50.982.00E−04 PMON80542 ZM_M57121 4.4 5.22 5 0.2467 −1.4 30.95 31.38 0.5865 945.99 42.21 0.0058 PMON80542 ZM_M57122 −3.5 5785.68 5993.37 0.1669 0.429.5 29.39 0.8458 8.9 55.51 50.98 1.00E−04 PMON80542 ZM_M57122 0 5 5 12.3 32.1 31.38 0.3537 6.8 45.07 42.21 0.0474 PMON80542 ZM_M57124 −35815.15 5993.37 0.2353 −2.7 28.61 29.39 0.1694 6.6 54.33 50.98 0.0032PMON80542 ZM_M57124 13.4 5.67 5 2.00E−04 0.3 31.48 31.38 0.8981 13.147.74 42.21 1.00E−04 PMON80542 ZM_M57131 13.3 7776.21 6866.4 0 6.5 33.5431.48 0.0099 27.9 68.11 53.23 0 PMON80542 ZM_M57132 −2.3 5853.25 5993.370.3506 −2.3 28.71 29.39 0.2306 13.2 57.73 50.98 0 PMON80542 ZM_M571327.6 5.38 5 0.0174 −1.9 30.79 31.38 0.4522 7.4 45.34 42.21 0.0221PMON80542 ZM_M57146 0.6 6031.47 5993.37 0.7995 4.9 30.82 29.39 0.0124−2.6 49.63 50.98 0.2347 PMON80542 ZM_M57146 0.4 5.02 5 0.9047 7 33.5831.38 0.0052 0.7 42.51 42.21 0.8221 123 PMON80850 ZM_M56061 −3.7 4.945.13 0.1027 2.8 30.43 29.6 0.2912 −1.8 44.81 45.62 0.6326 PMON80850ZM_M56061 −1.1 5272.7 5331.51 0.7088 0.7 28.56 28.35 0.7113 −6.3 42.3345.16 0.0465 PMON80850 ZM_M56062 3.5 5.31 5.13 0.1181 0.1 29.63 29.60.9654 6.2 48.44 45.62 0.0972 PMON80850 ZM_M56062 4.4 5566.18 5331.510.1369 1.6 28.8 28.35 0.4251 9.3 49.37 45.16 0.0032 PMON80850 ZM_M56071−3.3 4.96 5.13 0.141 2.5 30.33 29.6 0.379 7.6 49.1 45.62 0.0407PMON80850 ZM_M56071 −0.5 5302.33 5331.51 0.853 −0.8 28.11 28.35 0.669710.7 50.01 45.16 7.00E−04 PMON80850 ZM_M56222 −0.8 5.09 5.13 0.719 6.131.41 29.6 0.0211 0.9 46.01 45.62 0.8177 PMON80850 ZM_M56222 4 5545.235331.51 0.1754 4.2 29.53 28.35 0.0367 −0.8 44.78 45.16 0.7867 PMON80850ZM_M56722 −1.8 5.05 5.13 0.4557 0.1 29.61 29.6 0.9841 −5.6 43.05 45.620.1295 PMON80850 ZM_M56722 0.9 5379.37 5331.51 0.7693 2.1 28.94 28.350.3101 2.8 46.42 45.16 0.3906 PMON80850 ZM_M56723 −4.1 4.92 5.13 0.0711−1.2 29.25 29.6 0.6582 −2.9 44.28 45.62 0.4536 PMON80850 ZM_M56723 8.35774.12 5331.51 0.0052 2.1 28.94 28.35 0.2947 2.6 46.33 45.16 0.4113PMON80850 ZM_M57056 7.2 5.51 5.13 0.0014 5 31.06 29.6 0.0623 11.5 50.8545.62 0.0022 PMON80850 ZM_M57056 2.6 5472.58 5331.51 0.3707 1.4 28.7528.35 0.4782 2.1 46.11 45.16 0.5035

Nitrogen Use Field Efficacy Assay

Level I. Transgenic plants provided by the present invention are plantedin field without any nitrogen source being applied. Transgenic plantsand control plants are grouped by genotype and construct with controlsarranged randomly within genotype blocks. Each type of transgenic plantsare tested by 3 replications and across 5 locations. Nitrogen levels inthe fields are analyzed in early April pre-planting by collecting 30sample soil cores from 0-24″ and 24 to 48″ soil layer. Soil samples areanalyzed for nitrate-nitrogen, phosphorus(P), Potassium(K), organicmatter and pH to provide baseline values. P, K and micronutrients areapplied based upon soil test recommendations.Level II. Transgenic plants provided by the present invention areplanted in field with three levels of nitrogen (N) fertilizer beingapplied, i.e. low level (0 N), medium level (80 lb/ac) and high level(180 lb/ac). Liquid 28% or 32% UAN (Urea, Ammonium Nitrogen) are used asthe N source and apply by broadcast boom and incorporate with a fieldcultivator with rear rolling basket in the same direction as intendedcrop rows. Although there is no N applied to the 0 N treatment the soilshould still be disturbed in the same fashion as the treated area.Transgenic plants and control plants are grouped by genotype andconstruct with controls arranged randomly within genotype blocks. Eachtype of transgenic plants is tested by 3 replications and across 4locations. Nitrogen levels in the fields are analyzed in early Aprilpre-planting by collecting 30 sample soil cores from 0-24″ and 24 to 48″soil layer. Soil samples are analyzed for nitrate-nitrogen,phosphorus(P), Potassium(K), organic matter and pH to provide baselinevalues. P, K and micronutrients are applied based upon soil testrecommendations.

TABLE 9 Genes increase seed yield in transgenic plants at differentnitrogen levels. PEP SEQ Transgenic Control Percent ID NO Phe ID GeneConstruct Event mean Mean change Pvalue I 108 PHE0001623_1734 maizePMON79709 ZM_M51983 137.5 124.76521 10.207 0.0908 magnesium transporter,mrs2-1-like 1 105 PHE0001376_1468 Corn Rubisco PMON75524 ZM_M47998 140.2124.76521 12.3711 0.0407 Activase 2 130 PHE0001111_1201 Yeast alaninePMON77895 ZM_M61017 140.3 124.76521 12.4512 0.0394 aminotransferase PEPTrans- Per- P- SEQ ID treat- genic Control cent val- NO Phe ID GeneConstruct ment event yield yield change ue II 114 PHE0002412_2512Ralstonia pMON75980 High ZM_M53398 159.7 142.45 10.801503 0.0621metallidurans glutamate decarboxylase PHE0002412_2512 Ralstonia LowZM_M53398 137.125 125.14298 8.7380273 0.0263 metallidurans glutamatedecarboxylase PHE0002412_2512 Ralstonia High ZM_M53392 202.575190.5333333 5.9443005 0.0833 metallidurans glutamate decarboxylase 118PHE0002492_2592 Arabidopsis pMON80461 High ZM_M53218 160.6 142.4511.30137 0.0498 E2F PHE0002492_2592 Arabidopsis High ZM_M53848 158.675142.45 10.225303 0.0792 E2F PHE0002492_2592 Arabidopsis Low ZM_M53848141.175 125.14298 11.356132 0.0031 E2F PHE0002492_2592 Arabidopsis MedZM_M53218 159.15 145.075 8.843858 0.0883 E2F 91 PHE0001017_1108 MADS box110 pMON73816 Low ZM_M37188 134.575 125.14298 7.008746 0.0798

B. Selection for Increased Yield

Many transgenic plants of this invention exhibit improved yield ascompared to a control plant. Improved yield can result from enhancedseed sink potential, i.e. the number and size of endosperm cells orkernels and/or enhanced sink strength, i.e. the rate of starchbiosynthesis. Sink potential can be established very early during kerneldevelopment, as endosperm cell number and size are determined within thefirst few days after pollination.

Much of the increase in corn yield of the past several decades hasresulted from an increase in planting density. During that period, cornyield has been increasing at a rate of 2.1 bushels/acre/year, but theplanting density has increased at a rate of 250 plants/acre/year. Acharacteristic of modern hybrid corn is the ability of these varietiesto be planted at high density. Many studies have shown that a higherthan current planting density should result in more biomass production,but current germplasm does not perform. well at these higher densities.One approach to increasing yield is to increase harvest index (HI), theproportion of biomass that is allocated to the kernel compared to totalbiomass, in high density plantings.

Effective yield selection of enhanced yielding transgenic corn eventsuses hybrid progeny of the transgenic event over multiple locations withplants grown under optimal production management practices, and maximumpest control. A useful target for improved yield is a 5% to 10% increasein yield as compared to yield produced by plants grown from seed for acontrol plant. Selection methods may be applied in multiple and diversegeographic locations, for example up to 16 or more locations, over oneor more plating seasons, for example at least two planting seasons tostatistically distinguish yield improvement from natural environmentaleffects. It is to plant multiple transgenic plants, positive andnegative control plants, and pollinator plants in standard plots, forexample 2 row plots, 20 feet long by 5 feet wide with 30 inches distancebetween rows and a 3 foot alley between ranges. Transgenic events can begrouped by recombinant DNA constructs with groups randomly placed in thefield. A pollinator plot of a high quality corn line is planted forevery two plots to allow open pollination when using male steriletransgenic events. A useful planting density is about 30,000plants/acre. High planting density is greater than 30,000 plants/acre,preferably about 40,000 plants/acre, more preferably about 42,000plants/acre, most preferably about 45,000 plants/acre. Surrogateindicators for yield improvement include source capacity (biomass),source output (sucrose and photosynthesis), sink components (kernelsize, ear size, starch in the seed), development (light response,height, density tolerance), maturity, early flowering trait andphysiological responses to high density planting, for example at 45,000plants per acre, for example as illustrated in Table 10 and 11.

TABLE 10 Timing Evaluation Description comments V2-3 Early stand Can betaken any time after germination and prior to removal of any plants.Pollen shed GDU to 50% shed GDU to 50% plants shedding 50% tassel.Silking GDU to 50% silk GDU to 50% plants showing silks. Maturity Plantheight Height from soil surface to 10 plants per plot - Yield flag leafattachment (inches). team assistance Maturity Ear height Height fromsoil surface to 10 plants per plot - Yield primary ear attachment node.team assistance Maturity Leaves above ear visual scores: erect, size,rolling Maturity Tassel size Visual scores +/− vs. WT Pre-Harvest FinalStand Final stand count prior to harvest, exclude tillers Pre-HarvestStalk lodging No. of stalks broken below the primary ear attachment.Exclude leaning tillers Pre-Harvest Root lodging No. of stalksleaning >45° angle from perpendicular. Pre-Harvest Stay green Afterphysiological maturity and when differences among genotypes are evident:Scale 1 (90-100% tissue green)-9 (0-19% tissue green). Harvest GrainYield Grain yield/plot (Shell weight)

TABLE 11 Timing Evaluation Description V8-V12 Chlorophyll V12-VT Earleaf area V15-15DAP Chl fluorescence V15-15DAP CER 15-25 DAPCarbohydrates sucrose, starch Pre-Harvest 1st internode diameterPre-Harvest Base 3 internode diameter Pre-Harvest Ear internode diameterMaturity Ear traits diameter, length, kernel number, kernel weight

Electron transport rates (ETR) and CO2 exchange rates (CER): ETR and CERwere measured with Li6400LCF (Licor, Lincoln, Nebr.) around V9-R1stages. Leaf chlorophyll fluorescence is a quick way to monitor thesource activity and was reported to be highly correlated with CO₂assimilation under varies conditions (Photosyn Research, 37: 89-102).The youngest fully expanded leaf or 2 leaves above the ear leaf wasmeasured with actinic light 1500 (with 10% blue light) micromol m⁻² s⁻¹,28° C., CO2 levels 450 ppm. Ten plants were measured in each event.There were 2 readings for each plant.

A hand-held chlorophyll meter SPAD-502 (Minolta-Japan) was used tomeasure the total chlorophyll level on live transgenic plants and thewild type counterparts a. Three trifoliates from each plant wereanalyzed, and each trifoliate were analyzed three times. Then 9 datapoints were averaged to obtain the chlorophyll level. The number ofanalyzed plants of each genotype ranged from 5 to 8.

TABLE 12 pep Witchita, KS Carrollton, IL SEQ Mean SPAD % p- Mean SPAD %p- ID construct vaule change value vaule change value 88 pMON68399ZM_M31143 64.8 2 0.5215 58.87 4 0.0507 ZM_M31143 64.8 2 0.5828 ZM_M3114664 1 0.7624 54 −1.14 0.0337 ZM_M31146 64 1 0.8319 ZM_M31147 67.3 60.0858 59.84 6 0.0665 ZM_M31147 67.3 6 0.105 ZM_M31152 66.6 5 0.156458.9 1 0.7965 ZM_M31152 66.6 5 0.1862 ZM_M31524 60.4 −5 0.2009 57.44 20.5839 ZM_M31524 60.4 −5 0.1734 ZM_M32356 61.9 −2 0.5386 59.36 −2 0.4308ZM_M32356 61.9 −3 0.4836 ZM_M34171 62.7 −1 0.7919 60.18 0 0.9203ZM_M34171 62.7 −1 0.7255 ZM_M38646 64.5 2 0.6164 59.89 3 0.3042ZM_M38646 64.5 1 0.6819 ZM_M38660 67.3 6 0.0836 62.35 7 0.004

TABLE 13 PEP SEQ % % ID Construct event n-trt n-ctr ETR-ctr ChangePvalue CER-ctr Change 105 PMON75524 ZM_M47998 20 40 141.3 3 0.001 45.7 7PMON75524 ZM_M48003 20 40 141.3 8 0.000 45.7 6 PMON75524 ZM_M48004 20 40141.3 −4 0.000 45.7 −8 PMON75524 ZM_M48005 20 40 141.3 2 0.008 45.7 4PMON75524 ZM_M48007 20 40 141.3 4 0.000 45.7 −3 PMON75524 ZM_M48010 2040 141.3 6 0.000 45.7 8 125 PMON81853 ZM_M70887 18 64 136.3 −3 0.29843.7 −5 PMON81853 ZM_M70888 22 64 136.3 15 0.000 43.7 15 PMON81853ZM_M70889 22 64 136.3 −23 0.000 43.7 −18 PMON81853 ZM_M70900 22 64 136.3−14 0.000 43.7 −14 PMON81853 ZM_M71630 16 64 136.3 9 0.005 43.7 5 102PMON80542 ZM_M57107 20 101 154.1 0 0.863 40.5 5 PMON80542 ZM_M57119 20101 154.1 3 0.002 40.5 5 PMON80542 ZM_M57120 20 101 154.1 −6 0.000 40.5−4 PMON80542 ZM_M57121 20 101 154.1 −5 0.000 40.5 −8 PMON80542 ZM_M5712220 101 154.1 10 0.000 40.5 19 PMON80542 ZM_M57124 20 101 154.1 1 0.51440.5 3 PMON80542 ZM_M57131 20 101 154.1 6 0.000 40.5 7 PMON80542ZM_M57132 20 101 154.1 9 0.000 40.5 11 PMON80542 ZM_M57146 20 101 154.19 0.000 40.5 13 PEP SEQ % % ID Construct Pvalue n-trt n-ctr ETR-ctrChange Pvalue CER-ctr Change Pvalue 105 PMON75524 0.000 10 42 153.7 −00.978 45.8 −2 0.067 PMON75524 0.000 10 42 153.7 1 0.414 45.8 4 0.001PMON75524 0.000 11 42 153.7 7 0.000 45.8 9 0.000 PMON75524 0.012 12 42153.7 3 0.004 45.8 5 0.000 PMON75524 0.052 11 42 153.7 1 0.498 45.8 −20.072 PMON75524 0.000 10 42 153.7 7 0.000 45.8 9 0.000 125 PMON818530.097 19 51 151.5 8 0.001 34.8 9 0.012 PMON81853 0.000 10 51 151.5 110.000 34.8 22 0.000 PMON81853 0.000 16 51 151.5 10 0.000 34.8 13 0.000PMON81853 0.000 21 51 151.5 1 0.666 34.8 −0 0.944 PMON81853 0.119 10 51151.5 12 0.000 34.8 22 0.000 102 PMON80542 0.084 9 40 131.7 16 0.00028.9 18 0.000 PMON80542 0.099 10 40 131.7 −1 0.691 28.9 −3 0.304PMON80542 0.112 10 40 131.7 18 0.000 28.9 15 0.000 PMON80542 0.003 12 40131.7 −9 0.000 28.9 −12 0.000 PMON80542 0.000 9 40 131.7 −3 0.126 28.9−5 0.080 PMON80542 0.204 11 40 131.7 20 0.000 28.9 27 0.000 PMON805420.017 10 40 131.7 −3 0.098 28.9 −3 0.276 PMON80542 0.000 11 40 131.7 −40.025 28.9 −3 0.191 PMON80542 0.000 10 40 131.7 8 0.000 28.9 5 0.062

When selecting for yield improvement a useful statistical measurementapproach comprises three components, i.e. modeling spatialautocorrelation of the test field separately for each location,adjusting traits of recombinant DNA events for spatial dependence foreach location, and conducting an across location analysis. The firststep in modeling spatial autocorrelation is estimating the covarianceparameters of the semivariogram. A spherical covariance model is assumedto model the spatial autocorrelation. Because of the size and nature ofthe trial, it is likely that the spatial autocorrelation may change.Therefore, anisotropy is also assumed along with spherical covariancestructure. The following set of equations describes the statistical formof the anisotropic spherical covariance model.

${{C\left( {h;\theta} \right)} = {{v\; {I\left( {h = 0} \right)}} + {{\sigma^{2}\left( {1 - {\frac{3}{2}h} + {\frac{1}{2}h^{3}}} \right)}{I\left( {h < 1} \right)}}}},$

where I(•) is the indicator function

${h = \sqrt{{\overset{.}{x}}^{2} + {\overset{.}{y}}^{2}}},$

and

{dot over (x)}=[cos(ρπ/180)(x ₁ −x ₂)−sin(ρπ/180)(y ₁ −y ₂)]/ω_(x)

{dot over (y)}=[sin(ρπ/180)(x ₁ −x ₂)+cos(ρπ/180)(y ₁ −y ₂)]/ω_(y)

where s₁=(x₁, y₁) are the spatial coordinates of one location ands₂=(x₂, y₂) are the spatial coordinates of the second location. Thereare 5 covariance parameters, θ=(ν, σ², ρ, ω_(n), ω_(j)), where ν is thenugget effect, σ² is the partial sill, ρ is a rotation in degreesclockwise from north, ω_(n) is a scaling parameter for the minor axisand ω_(j) is a scaling parameter for the major axis of an anisotropicalellipse of equal covariance. The five covariance parameters that definesthe spatial trend will then be estimated by using data from heavilyreplicated pollinator plots via restricted maximum likelihood approach.In a multi-location field trial, spatial trend are modeled separatelyfor each location.

After obtaining the variance parameters of the model, avariance-covariance structure is generated for the data set to beanalyzed. This variance-covariance structure contains spatialinformation required to adjust yield data for spatial dependence. Inthis case, a nested model that best represents the treatment andexperimental design of the study is used along with thevariance-covariance structure to adjust the yield data. During thisprocess the nursery or the seed batch effects can also be modeled andestimated to adjust the yields for any yield parity caused by seed batchdifferences. After spatially adjusted data from different locations aregenerated, all adjusted data is combined and analyzed assuming locationsas replications. In this analysis, intra and inter-location variancesare combined to estimate the standard error of yield from transgenicplants and control plants. Relative mean comparisons are used toindicate statistically significant yield improvements.

TABLE 14 PEP SEQ construct Transgenic Mean Control Percent P- ID NO idevent control Transgenic mean difference value 105 pMON75524 ZM_M47998Negative 173.3 176.1 −1.6 0.392 segregant ZM_M48003 Negative 167.2 176.1−5.1 0.007 segregant ZM_M48004 Negative 176.2 176.1 0.0 0.990 segregantZM_M48005 Negative 186.0 176.1 5.6 0.003 segregant ZM_M48007 Negative177.9 176.1 1.0 0.631 segregant ZM_M48010 Negative 176.8 176.1 0.4 0.841segregant 88 pMON68399 ZM_M31146 Negative 179.1 179.9 −0.4 0.778segregant ZM_M31147 Negative 181.7 179.9 1.0 0.497 segregant ZM_M31524Negative 179.3 179.9 −0.3 0.829 segregant ZM_M32356 Negative 181.3 179.90.8 0.601 segregant ZM_M38646 Negative 180.3 179.9 0.2 0.880 segregantZM_M38681 Negative 180.2 179.9 0.2 0.894 segregant ZM_M39295 Negative176.6 179.9 −1.8 0.259 segregant ZM_M39297 Negative 175.6 179.9 −2.30.125 segregant ZM_M39298 Negative 184.6 179.9 2.7 0.082 segregantZM_M39302 Negative 182.0 179.9 1.2 0.440 segregant 105 pMON75524ZM_M47998 Negative 173.3 176.1 −1.6 0.392 segregant ZM_M48003 Negative167.2 176.1 −5.1 0.007 segregant ZM_M48004 Negative 176.2 176.1 0.00.990 segregant ZM_M48005 Negative 186.0 176.1 5.6 0.003 segregantZM_M48007 Negative 177.9 176.1 1.0 0.631 segregant ZM_M48010 Negative176.8 176.1 0.4 0.841 segregant 88 pMON68399 ZM_M31146 Negative 179.1179.9 −0.4 0.778 segregant ZM_M31147 Negative 181.7 179.9 1.0 0.497segregant ZM_M31524 Negative 179.3 179.9 −0.3 0.829 segregant ZM_M32356Negative 181.3 179.9 0.8 0.601 segregant ZM_M38646 Negative 180.3 179.90.2 0.880 segregant ZM_M38681 Negative 180.2 179.9 0.2 0.894 segregantZM_M39295 Negative 176.6 179.9 −1.8 0.259 segregant ZM_M39297 Negative175.6 179.9 −2.3 0.125 segregant ZM_M39298 Negative 184.6 179.9 2.70.082 segregant ZM_M39302 Negative 182.0 179.9 1.2 0.440 segregant

TABLE 15 Mean Mean Percent Construct Event Transgenic Control changeP-value PEP SEQ ID 127 PMON78911 ZM_M45101 167.9 176.1 −4.7 0.015 127PMON78911 ZM_M59413 175.4 176.1 −0.4 0.832 127 PMON78911 ZM_M59778 161.2176.1 −8.5 0.000 127 PMON78911 ZM_M59783 191.0 176.1 8.4 0.000 127PMON78911 ZM_M59784 182.6 176.1 3.7 0.053 127 PMON78911 ZM_M62810 180.2176.1 2.3 0.212 130 PMON77895 ZM_M61016 171.5 176.1 −2.6 0.163 139PMON77895 ZM_M61017 173.4 176.1 −1.6 0.397 130 PMON77895 ZM_M61033 184.1176.1 4.5 0.015 131 PMON79152 ZM_M64367 162.9 176.1 −7.5 0.000 131PMON79152 ZM_M65978 184.5 176.1 4.7 0.012 131 PMON79152 ZM_M65982 175.0176.1 −0.6 0.733 131 PMON79152 ZM_M65986 139.7 176.1 −20.7 0.000 131PMON79152 ZM_M65992 171.8 176.1 −2.5 0.182 132 PMON80921 ZM_M63833 184.2176.1 4.6 0.015 133 PMON75505 ZM_M49384 183.6 176.1 4.2 0.023 134PMON80925 ZM_M60505 183.4 176.1 4.1 0.039 134 PMON80925 ZM_M62005 179.8176.1 2.1 0.268 134 PMON80925 ZM_M62007 178.5 176.1 1.3 0.489 134PMON80925 ZM_M63594 180.1 176.1 2.3 0.229 106 PMON79163 ZM_M45011 177.0176.1 0.5 0.792 106 PMON79163 ZM_M48217 179.8 176.1 2.1 0.289 106PMON79163 ZM_M61816 183.5 176.1 4.2 0.033 106 PMON79163 ZM_M61822 168.1176.1 −4.6 0.023 136 PMON79164 ZM_M44045 172.1 176.1 −2.3 0.217 136PMON79164 ZM_M59749 180.6 176.1 2.5 0.175 136 PMON79164 ZM_M59750 181.8176.1 3.2 0.087 136 PMON79164 ZM_M61349 169.5 176.1 −3.8 0.042 136PMON79164 ZM_M61889 175.0 176.1 −0.6 0.738 136 PMON79164 ZM_M61890 145.4176.1 −17.4 0.000 136 PMON79164 ZM_M62988 175.7 176.1 −0.3 0.881 136PMON79164 ZM_M63003 185.0 176.1 5.0 0.007 107 PMON75533 ZM_M47453 183.4176.1 4.1 0.027 107 PMON75533 ZM_M47460 178.4 176.1 1.3 0.491 107PMON75533 ZM_M49275 183.9 176.1 4.4 0.018 107 PMON75533 ZM_M49278 177.0176.1 0.5 0.790 137 PMON79653 ZM_M49883 174.6 176.1 −0.9 0.633 137PMON79653 ZM_M65281 183.4 176.1 4.1 0.030 138 PMON81228 ZM_M59931 169.3176.1 −3.9 0.055 138 PMON81228 ZM_M60825 185.8 176.1 5.5 0.003 148PMON82223 ZM_M70571 185.8 176.1 5.5 0.007 161 PMON79665 ZM_M51224 171.9176.1 −2.4 0.198 161 PMON79665 ZM_M53787 172.2 176.1 −2.2 0.233 161PMON79665 ZM_M55078 184.2 176.1 4.6 0.019 139 PMON79430 ZM_M50221 181.1176.1 2.8 0.137 139 PMON79430 ZM_M50222 178.6 176.1 1.4 0.477 139PMON79430 ZM_M50223 180.8 176.1 2.7 0.153 139 PMON79430 ZM_M50727 177.7176.1 0.9 0.637 139 PMON79430 ZM_M50729 179.0 176.1 1.6 0.377 139PMON79430 ZM_M51479 171.7 176.1 −2.5 0.198 139 PMON79430 ZM_M51481 185.4176.1 5.2 0.006 139 PMON79430 ZM_M51490 178.5 176.1 1.3 0.492 140PMON79731 ZM_M52239 187.5 176.1 6.5 0.001 140 PMON79731 ZM_M52245 172.2176.1 −2.2 0.230 140 PMON79731 ZM_M52252 174.6 176.1 −0.9 0.638 140PMON79731 ZM_M52255 172.4 176.1 −2.1 0.248 140 PMON79731 ZM_M52375 173.3176.1 −1.6 0.396 140 PMON79731 ZM_M52802 173.6 176.1 −1.5 0.447 140PMON79731 ZM_M52812 166.6 176.1 −5.4 0.004 141 PMON78229 ZM_M55961 176.0176.1 −0.1 0.963 141 PMON78229 ZM_M55962 182.3 176.1 3.5 0.065 141PMON78229 ZM_M55964 175.1 176.1 −0.6 0.743 141 PMON78229 ZM_M56184 187.2176.1 6.3 0.001 141 PMON78229 ZM_M56185 181.8 176.1 3.2 0.083 141PMON78229 ZM_M59082 176.1 176.1 0.0 0.984 SEQ ID NO 116 PMON79697ZM_M53938 171.6 176.1 −2.6 0.171 116 PMON79697 ZM_M53939 180.2 176.1 2.30.238 116 PMON79697 ZM_M54371 175.0 176.1 −0.6 0.733 116 PMON79697ZM_M54372 185.1 176.1 5.1 0.009 116 PMON79697 ZM_M54374 181.2 176.1 2.80.127 144 PMON78240 ZM_M53464 184.1 176.1 4.5 0.015 144 PMON78240ZM_M53465 175.2 176.1 −0.5 0.785 144 PMON78240 ZM_M53470 174.4 176.1−1.0 0.611 144 PMON78240 ZM_M53471 166.7 176.1 −5.4 0.005 144 PMON78240ZM_M53478 173.6 176.1 −1.4 0.456 144 PMON78240 ZM_M53673 175.8 176.1−0.2 0.917 144 PMON78240 ZM_M53674 172.5 176.1 −2.1 0.269 144 PMON78240ZM_M53684 179.4 176.1 1.8 0.342 122 PMON80500 ZM_M56549 173.4 176.1 −1.60.408 122 PMON80500 ZM_M56560 173.4 176.1 −1.6 0.394 122 PMON80500ZM_M56565 175.4 176.1 −0.4 0.811 122 PMON80500 ZM_M56567 177.9 176.1 1.00.599 122 PMON80500 ZM_M56568 185.9 176.1 5.6 0.003 122 PMON80500ZM_M58003 169.4 176.1 −3.8 0.047 145 PMON80283 ZM_M58140 174.6 176.1−0.9 0.641 145 PMON80283 ZM_M58141 179.7 176.1 2.0 0.294 145 PMON80283ZM_M58143 183.8 176.1 4.4 0.024 146 PMON80866 ZM_M58256 177.6 176.1 0.80.651 146 PMON80866 ZM_M59441 183.3 176.1 4.1 0.028 146 PMON80866ZM_M60646 174.8 176.1 −0.7 0.692 147 PMON80292 ZM_M57487 180.8 176.1 2.60.159 147 PMON80292 ZM_M58571 184.2 176.1 4.6 0.021 147 PMON80292ZM_M58578 177.5 176.1 0.8 0.717 142 PMON79696 ZM_M53849 177.6 179.1 −1.20.431 142 PMON79696 ZM_M53849 190.3 179.1 5.8 0.0003 142 PMON79696ZM_M53849 178.5 179.1 −0.7 0.0635 150 PMON81857 ZM_M67504 178.8 176.11.5 0.415 150 PMON81857 ZM_M70000 182.7 176.1 3.7 0.047 150 PMON81857ZM_M71064 172.1 176.1 −2.3 0.229 150 PMON81857 ZM_M71065 184.6 176.1 4.80.011 150 PMON81857 ZM_M72550 174.3 176.1 −1.0 0.589 149 PMON83553ZM_M71131 150.7 176.1 −14.5 0.000 149 PMON83553 ZM_M71140 187.4 176.16.4 0.001 149 PMON83553 ZM_M71156 150.3 176.1 −14.7 0.000 149 PMON83553ZM_M71161 172.7 176.1 −1.9 0.298 150 PMON81857 ZM_M67504 178.8 176.1 1.50.415 150 PMON81857 ZM_M70000 182.7 176.1 3.7 0.047 150 PMON81857ZM_M71064 172.1 176.1 −2.3 0.229 150 PMON81857 ZM_M71065 184.6 176.1 4.80.011 150 PMON81857 ZM_M72550 174.3 176.1 −1.0 0.589 151 PMON82212ZM_M67581 171.1 176.1 −2.8 0.126 151 PMON82212 ZM_M67583 186.1 176.1 5.60.002 151 PMON82212 ZM_M69111 173.2 176.1 −1.7 0.368 PEP SEQ ID NO 108PMON79709 ZM_M51983 184.3 176.1 4.7 0.037 108 PMON79709 ZM_M51985 180.1176.1 2.3 0.231 108 PMON79709 ZM_M52052 185.6 176.1 5.3 0.013 108PMON79709 ZM_M52710 175.5 176.1 −0.4 0.862 108 PMON79709 ZM_M52720 175.2176.1 −0.6 0.765 129 PMON73787 ZM_M55089 162.6 176.1 −7.7 0.000 129PMON73787 ZM_M61950 186.4 176.1 5.8 0.002 129 PMON73787 ZM_M61953 164.7176.1 −6.5 0.001 129 PMON73787 ZM_M61958 165.9 176.1 −5.8 0.003 129PMON73787 ZM_M61965 134.3 176.1 −23.8 0.000 129 PMON73787 ZM_M61966172.6 176.1 −2.0 0.280 135 PMON78942 ZM_M66312 176.2 176.1 0.0 0.997 135PMON78942 ZM_M66316 173.1 176.1 −1.7 0.362 135 PMON78942 ZM_M66318 164.1176.1 −6.9 0.000 135 PMON78942 ZM_M66331 183.3 176.1 4.1 0.029

C. Selection for Enhanced Water Use Efficiency (WUE)

Described in this example is a high-throughput method for greenhouseselection of transgenic corn plants to wild type corn plants (tested asinbreds or hybrids) for water use efficiency. This selection processimposes 3 drought/re-water cycles on plants over a total period of 15days after an initial stress free growth period of 11 days. Each cycleconsists of 5 days, with no water being applied for the first four daysand a water quenching on the 5th day of the cycle. The primaryphenotypes analyzed by the selection method are the changes in plantgrowth rate as determined by height and biomass during a vegetativedrought treatment. The hydration status of the shoot tissues followingthe drought is also measured. The plant height are measured at threetime points. The first is taken just prior to the onset drought when theplant is 11 days old, which is the shoot initial height (SIH). The plantheight is also measured halfway throughout the drought/re-water regimen,on day 18 after planting, to give rise to the shoot mid-drought height(SMH). Upon the completion of the final drought cycle on day 26 afterplanting, the shoot portion of the plant is harvested and measured for afinal height, which is the shoot wilt height (SWH) and also measured forshoot wilted biomass (SWM). The shoot is placed in water at 40 degreeCelsius in the dark. Three days later, the shoot is weighted to giverise to the shoot turgid weight (STM). After drying in an oven for fourdays, the shoots are weighted for shoot dry biomass (SDM). The shootaverage height (SAH) is the mean plant height across the 3 heightmeasurements. The procedure described above may be adjusted for +/−˜oneday for each step given the situation.

To correct for slight differences between plants, a size correctedgrowth value is derived from SIH and SWH. This is the Relative GrowthRate (RGR). Relative Growth Rate (RGR) is calculated for each shootusing the formula [RGR %=(SWH−SIH)/((SWH+SIH)/2)*100]. Relative watercontent (RWC) is a measurement of how much (%) of the plant was water atharvest. Water Content (RWC) is calculated for each shoot using theformula [RWC %=(SWM−SDM)/(STM−SDM)*100]. Fully watered corn plants ofthis age run around 98% RWC.

The transgenic plants provided by this invention were selected throughthe selection process according to the standard procedure describedabove and the performance of these transgenic plants are shown in Table16 below.

TABLE 16 PEP SEQ N Perc, Pvalue, Perc, Pvalue, Perc, Pvalue, Perc,Pvalue, ID NO Construct Event SAH SAH RGR RGR SDM SDM RWC RWC 88PMON68399 18 −2.9129 0 4.6104 0 −1.2282 0.0534 2.0799 0 87 PMON72494 2−2.6854 0 3.3347 0.0034 −3.178 0.0258 2.8177 0.0001 PMON72494 2 −1.41890 4.5389 0 1.0503 0.2808 1.8075 0.0272 PMON72494 6 −2.8912 0 5.0217 0−3.0056 0.0032 3.0684 0 PMON72494 1 −3.2736 0 1.4026 0.2741 0.09680.9545 −2.3654 0.0194 97 PMON76342 1 −3.6096 0.0003 8.9657 0 −2.93320.2317 2.1037 0.1252 PMON76342 2 −0.9997 0.0384 4.9006 0 −1.7424 0.1472−0.8155 0.2552 117 PMON78237 4 −2.0513 0 2.1335 0.0011 3.2477 0.00020.5998 0.2456 104 PMON78936 2 0.2781 0.3727 1.3631 0.0165 2.1849 0.0231.4237 0.0744 PMON78936 4 −2.3342 0 6.1784 0 −2.5964 0.0336 2.53580.0003 103 PMON78949 4 −1.6398 0 4.5323 0 2.2077 0.0112 0.9068 0.08 109PMON79422 4 −2.0016 0 2.8698 0 −1.3511 0.0488 1.8883 0.0009 116PMON79697 2 −1.0829 0.1252 2.9806 0.0225 −0.0495 0.9771 0.0115 0.9907PMON79697 3 −1.5704 0 2.1663 0 −0.4949 0.5582 1.7787 0.0073 120PMON80452 1 −1.7626 0.0032 2.1476 0.2778 2.1702 0.3832 −1.914 0.1164PMON80452 8 −0.2756 0.0645 −1.0206 0.0002 0.4707 0.3101 −0.072 0.8521PMON80452 11 −0.7077 0.0258 2.1403 0.0003 1.4477 0.0623 −0.0405 0.9267115 PMON80489 6 −0.895 0.0001 3.7262 0 −1.5941 0.0442 1.4212 0.0038 102PMON80542 8 −2.5925 0 1.1234 0.0254 2.1829 0.0013 3.2415 0 PMON80542 1−5.5931 0 2.5902 0.0486 −2.1444 0.2158 9.5238 0

Transgenic plants transformed with pMON67754 comprising the recombinantDNA as set forth in SEQ ID NO: 3 were tested in field with moderatedrought conditions in Satanta, Ill. and Dixon Calif. SPAD readings onleaves under a moderate drought stress showed a significant increase inchlorophyll level in the transgenic plants as compared to the controlplants. Two events showed a significant increase in SPAD reading forchlorophyll level, indicating an improvement in drought tolerance. Inreplicated field trials, 2 events (ZM_M16396 and ZM_M16401) out of 6tested, showed significantly (p<0.1) improved leaf SPAD readings in twodifferent locations, indicating an improvement in drought tolerance.

D. Selection for Growth Under Cold Stress

(1) Cold germination assay—Three sets of seeds are used for the assay.The first set consists of positive transgenic events (F1 hybrid) wherethe genes of the present invention are expressed in the seed. The secondseed set is nontransgenic, wild-type negative control made from the samegenotype as the transgenic events. The third set consisted of two coldtolerant and one cold sensitive commercial check lines of corn. Allseeds are treated with a fungicide “Captan” (MAESTRO® 80DF Fungicide,Arvesta Corporation, San Francisco, Calif., USA). 0.43 mL Captan isapplied per 45 g of corn seeds by mixing it well and drying thefungicide prior to the experiment.

Corn kernels are placed embryo side down on blotter paper within anindividual cell (8.9×8.9 cm) of a germination tray (54×36 cm). Ten seedsfrom an event are placed into one cell of the germination tray. Eachtray can hold 21 transgenic events and 3 replicates of wildtype(LH244SDms+LH59), which is randomized in a complete block design. Forevery event there are five replications (five trays). The trays areplaced at 9.7 C for 24 days (no light) in a Convrion growth chamber(Conviron Model PGV36, Controlled Environments, Winnipeg, Canada). Twohundred and fifty millilters of deionized water are added to eachgermination tray. Germination counts are taken 10th, 11th, 12th, 13th,14th, 17th, 19th, 21st, and 24th day after start date of the experiment.Seeds are considered germinated if the emerged radicle size is 1 cm.From the germination counts germination index is calculated.

The germination index is calculated as per:

Germination index=(Σ([T+1+−n _(i) ]*[P _(i) −P _(i-1)]))/T

Where T is the total number of days for which the germination assay isperformed. The number of days after planting is defined by n. “i”indicated the number of times the germination had been counted,including the current day. P is the percentage of seeds germinatedduring any given rating. Statistical differences are calculated betweentransgenic events and wild type control. After statistical analysis, theevents that show a statistical significance at the p level of less than0.1 relative to wild-type controls will advance to a secondary coldselection. The secondary cold screen is conducted in the same manner ofthe primary selection only increasing the number of repetitions to ten.Statistical analysis of the data from the secondary selection isconducted to identify the events that show a statistical significance atthe p level of less than 0.05 relative to wild-type controls.

TABLE 17 Germination index PEP SEQ Percent Mean of ID Construct ID EventID change Mean controls P-value 85 PMON69456 ZM_M15392 −27 23.4 32.070.0718 PMON69456 ZM_M15392 12 47.88 42.93 9.00E−04 PMON69456 ZM_M1539213 48 42.44 0.0756 PMON69456 ZM_M17042 −9 29.2 32.07 0.4 PMON69456ZM_M17042 17 49.5 42.44 0.0248 PMON69456 ZM_M17042 16 49.89 42.93 0PMON69456 ZM_M17042 −6 28.14 30.07 0.6526 PMON69456 ZM_M17044 −38 19.2530.88 0.019 PMON69456 ZM_M17044 9 46.17 42.44 0.2317 PMON69456 ZM_M170447 46.88 43.86 0.0297 PMON69456 ZM_M17044 14 34.14 30.07 0.3445 107PMON75533 ZM_M47453 3 46.88 45.38 0.3782 PMON75533 ZM_M47453 25 49.7539.69 0.002 PMON75533 ZM_M47460 23 48.83 39.69 0.0047 PMON75533ZM_M47460 3 46.88 45.38 0.3782 PMON75533 ZM_M49275 14 45.08 39.69 0.0914PMON75533 ZM_M49275 11 50.46 45.38 0.0031 PMON75533 ZM_M49278 15 45.8339.69 0.055 PMON75533 ZM_M49278 14 51.75 45.38 2.00E−04 119 PMON78235ZM_M53641 16 48.25 41.72 4.00E−04 PMON78235 ZM_M53641 23 45 36.5 0.0508PMON78235 ZM_M53641 1 48.42 48.08 0.9116 PMON78235 ZM_M53641 5 42.1740.24 0.5629 PMON78235 ZM_M53994 26 46 36.5 0.0294 PMON78235 ZM_M5399415 47.92 41.72 7.00E−04 PMON78235 ZM_M53994 1 48.67 48.08 0.8459PMON78235 ZM_M53994 −4 38.58 40.24 0.6196 PMON78235 ZM_M53997 16 48.2141.72 4.00E−04 PMON78235 ZM_M53997 15 42 36.5 0.2036 104 PMON78936ZM_M45248 25 48.25 38.69 0.0221 PMON78936 ZM_M45248 14 48.29 42.210.0013 PMON78936 ZM_M45274 15 48.33 42.21 0.0012 PMON78936 ZM_M45274 2448.08 38.69 0.0245 PMON78936 ZM_M45275 5 40.5 38.69 0.6613 PMON78936ZM_M46485 11 42.92 38.69 0.3066 PMON78936 ZM_M46516 −1 38.33 38.690.9301 PMON78936 ZM_M46516 −4 40.38 42.21 0.3274 PMON78936 ZM_M47276 1143.08 38.69 0.288 110 PMON79425 ZM_M50823 4 42.79 41.31 0.3848 PMON79425ZM_M50823 18 42.83 36.25 0.0378 PMON79425 ZM_M50856 4 42.88 41.31 0.3589PMON79425 ZM_M50856 13 40.83 36.25 0.1462 PMON79425 ZM_M51300 7 44.2541.31 0.087 PMON79425 ZM_M51300 −3 35.16 36.25 0.7282 PMON79425ZM_M51302 23 44.54 36.25 0.0093 PMON79425 ZM_M51302 17 48.17 41.311.00E−04 PMON79425 ZM_M51313 12 46.33 41.31 0.004 PMON79425 ZM_M51313 2344.7 36.25 0.008 PMON79425 ZM_M51608 24 45.08 36.25 0.0057 PMON79425ZM_M51608 11 45.88 41.31 0.0086 PMON79425 ZM_M51623 21 43.7 36.25 0.0189PMON79425 ZM_M51623 14 47.21 41.31 8.00E−04 PMON79425 ZM_M52067 −5 39.1341.31 0.2033 PMON79425 ZM_M52067 8 39.08 36.25 0.368 116 PMON79697ZM_M53938 7 47.04 43.93 0.0587 PMON79697 ZM_M53938 5 42 40.17 0.6198PMON79697 ZM_M53939 18 47.25 40.17 0.0575 PMON79697 ZM_M53939 11 48.5843.93 0.0049 PMON79697 ZM_M54371 11 48.88 43.93 0.0028 PMON79697ZM_M54371 15 46.25 40.17 0.1019 PMON79697 ZM_M54372 1 40.75 40.17 0.8745PMON79697 ZM_M54374 12 49.21 43.93 0.0022 PMON79697 ZM_M54374 18 47.2540.17 0.0575 111 PMON79718 ZM_M50838 6 45.25 42.78 0.331 PMON79718ZM_M51591 −3 42.67 43.93 0.4409 PMON79718 ZM_M51591 −18 35.08 42.780.0031 PMON79718 ZM_M51592 −3 41.42 42.78 0.5919 PMON79718 ZM_M51594 646.46 43.93 0.1241 PMON79718 ZM_M51594 13 48.15 42.78 0.0545 PMON79718ZM_M51598 11 48.96 43.93 0.0024 PMON79718 ZM_M51598 11 47.58 42.780.0606 PMON79718 ZM_M51615 6 46.46 43.93 0.1241 PMON79718 ZM_M51615 1147.33 42.78 0.075 PMON79718 ZM_M51618 2 43.5 42.78 0.7759 PMON79718ZM_M52797 −6 40.17 42.78 0.3047 PMON79718 ZM_M52937 16 49.67 42.780.0077 PMON79718 ZM_M52937 12 49.04 43.93 0.0021 96 PMON80270 ZM_M5596710.19 50.63 45.94 6.00E−04 PMON80270 ZM_M55968 7.38 49.33 45.94 0.0129PMON80270 ZM_M55969 3.27 47.44 45.94 0.2678 PMON80270 ZM_M55970 10.5650.79 45.94 4.00E−04 PMON80270 ZM_M55971 7.38 49.33 45.94 0.0129PMON80270 ZM_M55972 2.66 47.17 45.94 0.3663 PMON80270 ZM_M56524 3.8147.7 45.94 0.1952 PMON80270 ZM_M56526 −7.6 42.46 45.94 0.0105 PMON80270ZM_M56527 −19.87 36.82 45.94 0 120 PMON80452 ZM_M53452 13 41.83 37.080.1902 PMON80452 ZM_M53452 19 49.63 41.56 0 PMON80452 ZM_M53452 7 51.4248.08 0.2683 PMON80452 ZM_M53452 0 40.25 40.24 0.9971 PMON80452ZM_M53455 −3 36 37.08 0.7642 PMON80452 ZM_M53455 17 48.67 41.56 0PMON80452 ZM_M53455 −9 43.67 48.08 0.1434 PMON80452 ZM_M53455 −1 39.9240.24 0.9231 PMON80452 ZM_M53456 18 49.17 41.56 0 PMON80452 ZM_M53456 1843.83 37.08 0.0639 PMON80452 ZM_M53469 14 47.54 41.56 1.00E−04 PMON80452ZM_M53469 18 43.75 37.08 0.0672 PMON80452 ZM_M53694 14 42.42 37.080.1418 PMON80452 ZM_M53694 13 46.92 41.56 4.00E−04 PMON80452 ZM_M5369521 50.08 41.56 0 PMON80452 ZM_M53695 22 45.25 37.08 0.0256 PMON80452ZM_M53696 21 50.42 41.56 0 PMON80452 ZM_M53696 31 48.5 37.08 0.002PMON80452 ZM_M54104 13 41.75 37.08 0.198 PMON80452 ZM_M54104 13 47.1741.56 2.00E−04 PMON80452 ZM_M54106 8 39.92 37.08 0.4332 PMON80452ZM_M54106 12 46.38 41.56 0.0015 118 PMON80461 ZM_M52932 17 48.67 41.56 0PMON80461 ZM_M52932 32 48.17 36.5 0.0079 PMON80461 ZM_M52932 −8 43.2546.86 0.1944 PMON80461 ZM_M52932 9 43.92 40.24 0.271 PMON80461 ZM_M5321816 42.42 36.5 0.1717 PMON80461 ZM_M53218 7 44.58 41.56 0.0448 PMON80461ZM_M53218 −6 44.08 46.86 0.3172 PMON80461 ZM_M53218 4 41.92 40.24 0.6145PMON80461 ZM_M53235 22 50.71 41.56 0 PMON80461 ZM_M53235 24 45.25 36.50.0445 PMON80461 ZM_M53503 13 46.79 41.56 6.00E−04 PMON80461 ZM_M5350328 46.83 36.5 0.0181 PMON80461 ZM_M53504 12 41 36.5 0.2975 PMON80461ZM_M53504 14 47.5 41.56 1.00E−04 PMON80461 ZM_M53848 24 51.57 41.56 0PMON80461 ZM_M53848 15 41.92 36.5 0.2104 PMON80461 ZM_M54282 22 50.7541.56 0 PMON80461 ZM_M54282 29 47 36.5 0.0164 PMON80461 ZM_M54284 2144.33 36.5 0.0714 PMON80461 ZM_M54284 22 50.71 41.56 0 PMON80461ZM_M55266 7 50.22 46.86 0.2268 PMON80461 ZM_M55957 10 51.53 46.86 0.0945PMON80461 ZM_M56233 9 51.18 46.86 0.1217 PMON80461 ZM_M56728 2 47.9246.86 0.7033 122 PMON80500 ZM_M56549 −0.52 45.71 45.94 0.8613 PMON80500ZM_M56560 8.29 49.75 45.94 0.0053 PMON80500 ZM_M56565 2.2 46.96 45.940.4535 PMON80500 ZM_M56567 9.19 50.17 45.94 0.002 PMON80500 ZM_M5656810.82 50.92 45.94 3.00E−04 PMON80500 ZM_M58003 4.2 47.88 45.94 0.1542(2) Cold Shock assay—The experimental set-up for the cold shock assaywas the same as described in the above cold germination assay exceptseeds were grown in potted media for the cold shock assay.

The desired numbers of 2.5″ square plastic pots were placed on flats(n=32, 4×8). Pots were filled with Metro Mix 200 soil-less mediacontaining 19:6:12 fertilizer (6 lbs/cubic yard) (Metro Mix, Pots andFlat are obtained from Hummert International, Earth City, Mo.). Afterplanting seeds, pots were placed in a growth chamber set at 23° C.,relative humidity of 65% with 12 hour day and night photoperiod (300uE/m2-min). Planted seeds were watered for 20 minute every other day bysub-irrigation and flats were rotated every third day in a growthchamber for growing corn seedlings.

On the 10^(th) day after planting the transgenic positive and wild-typenegative (WT) plants were positioned in flats in an alternating pattern.Chlorophyll fluorescence of plants was measured on the 10^(th) dayduring the dark period of growth by using a PAM-2000 portablefluorometer as per the manufacturer's instructions (Walz, Germany).After chlorophyll measurements, leaf samples from each event werecollected for confirming the expression of genes of the presentinvention. For expression analysis six V1 leaf tips from each selectionwere randomly harvested. The flats were moved to a growth chamber set at5° C. All other conditions such as humidity, day/night cycle and lightintensity were held constant in the growth chamber. The flats weresub-irrigated every day after transfer to the cold temperature. On the4^(th) day chlorophyll fluorescence was measured. Plants weretransferred to normal growth conditions after six days of cold shocktreatment and allowed to recover for the next three days. During thisrecovery period the length of the V3 leaf was measured on the 1^(st) and3^(rd) days. After two days of recovery V2 leaf damage was determinedvisually by estimating percent of green V2 leaf.

Statistical differences in V3 leaf growth, V2 leaf necrosis andfluorescence during pre-shock and cold shock can be used for estimationof cold shock damage on corn plants.

(3) Early seedling growth assay—Three sets of seeds were used for theexperiment. The first set consists of positive transgenic events (F1hybrid) where the genes of the present invention were expressed in theseed. The second seed set was nontransgenic, wild-type negative controlmade from the same genotype as the transgenic events. The third seed setconsisted of two cold tolerant and two cold sensitive commercial checklines of corn. All seeds were treated with a fungicide “Captan”,(3a,4,7,a-tetrahydro-2-[(trichloromethly)thio]-1H-isoindole-1,3(2H)-dione,Drex Chemical Co. Memphis, Tenn.). Captan (0.43 mL) was applied per 45 gof corn seeds by mixing it well and drying the fungicide prior to theexperiment.

Seeds were grown in germination paper for the early seedling growthassay. Three 12″×18″ pieces of germination paper (Anchor Paper #SD7606)were used for each entry in the test (three repetitions per transgenicevent). The papers were wetted in a solution of 0.5% KNO₃ and 0.1%Thyram.

For each paper fifteen seeds were placed on the line evenly spaced downthe length of the paper. The fifteen seeds were positioned on the papersuch that the radical would grow downward, for example longer distanceto the paper's edge. The wet paper was rolled up starting from one ofthe short ends. The paper was rolled evenly and tight enough to hold theseeds in place. The roll was secured into place with two large paperclips, one at the top and one at the bottom. The rolls were incubated ina growth chamber at 23° C. for three days in a randomized complete blockdesign within an appropriate container. The chamber was set for 65%humidity with no light cycle. For the cold stress treatment the rollswere then incubated in a growth chamber at 12° C. for twelve days. Thechamber was set for 65% humidity with no light cycle.

After the cold treatment the germination papers were unrolled and theseeds that did not germinate were discarded. The lengths of the radicleand coleoptile for each seed were measured through an automated imagingprogram that automatically collects and processes the images. Theimaging program automatically measures the shoot length, root length,and whole seedling length of every individual seedling and thencalculates the average of each roll.

After statistical analysis, the events that show a statisticalsignificance at the p level of less than 0.1 relative to wild-typecontrols will advance to a secondary cold selection. The secondary coldselection is conducted in the same manner of the primary selection onlyincreasing the number of repetitions to five. Statistical analysis ofthe data from the secondary selection is conducted to identify theevents that show a statistical significance at the p level of less than0.05 relative to wild-type controls.

TABLE 18 Root length Shoot length Seedlling length Mean Mean Mean PEP ofP- of P- of P- SEQ Construct Event Percent con- val- Percent con- val-Percent con- val- ID ID ID change Mean trols ue change Mean trols uechange Mean trols ue 88 PMON68399 ZM_M31143 −4 9.5 9.85 0.5479 −1 7.946.04 0.7666 −3 17.45 17.9 0.6024 PMON68399 ZM_M31143 7 11.15 10.410.1158 8 9.42 8.69 0.0536 8 20.57 19.1 0.0607 PMON68399 ZM_M31146 1111.52 10.41 0.0186 0 8.67 8.69 0.9668 6 20.2 19.1 0.1593 PMON68399ZM_M31146 14 9.9 8.66 0.011 6 7.37 6.99 0.2969 10 17.27 15.65 0.0396PMON68399 ZM_M31147 13 11.75 10.41 0.0051 12 9.69 8.69 0.0088 12 21.4319.1 0.0034 PMON68399 ZM_M31147 14 11.25 9.85 0.0185 4 8.33 8.04 0.39619 19.58 17.9 0.0513 PMON68399 ZM_M31152 −20 8.4 10.45 2.00E−04 −7 7.447.96 0.3265 −14 15.84 18.41 0.0087 PMON68399 ZM_M31152 1 10.48 10.410.8793 6 9.17 8.69 0.1965 3 19.66 19.1 0.4697 PMON68399 ZM_M31524 1512.01 10.41 9.00E−04 10 9.54 8.69 0.0242 13 21.55 19.1 0.0021 PMON68399ZM_M31524 12 11.08 9.85 0.0385 8 8.69 8.04 0.0569 11 19.77 17.9 0.0306PMON68399 ZM_M32356 12 10.99 9.85 0.0533 −1 7.99 8.04 0.8731 6 18.9817.9 0.2052 PMON68399 ZM_M32356 12 11.7 10.41 0.0068 7 9.32 8.69 0.09610 21.01 19.1 0.0153 PMON68399 ZM_M34171 −24 8.6 11.39 4.00E−04 −13 7.358.48 0.0331 −20 15.95 19.87 0.0016 PMON68399 ZM_M34171 13 11.72 10.410.006 6 9.23 8.69 0.1486 10 20.95 19.1 0.0187 PMON68399 ZM_M38646 1012.63 11.52 0.032 3 10.38 10.05 0.4864 7 23.01 21.57 0.106 PMON68399ZM_M38660 10 12.68 11.52 0.0249 3 10.37 10.05 0.4953 7 23.06 21.570.0947 PMON68399 ZM_M38681 6 12.2 11.52 0.1829 3 10.31 10.05 0.5738 422.52 21.57 0.2835 PMON68399 ZM_M38697 7 12.35 11.52 0.1053 0 10.0310.05 0.9751 4 22.38 21.57 0.3563 PMON68399 ZM_M39295 11 12.84 11.520.0115 11 11.12 10.05 0.0264 11 23.97 21.57 0.0084 PMON68399 ZM_M3929720 13.84 11.52 0 7 10.79 10.05 0.1203 14 24.63 21.57 0.001 PMON68399ZM_M39298 7 12.29 11.52 0.1342 −1 9.91 10.05 0.7669 3 22.19 21.57 0.4785PMON68399 ZM_M39299 6 12.17 11.52 0.2051 1 10.13 10.05 0.8674 3 22.2921.57 0.4118 PMON68399 ZM_M39302 −44 6.44 11.52 0 −31 6.98 10.05 0 −3813.42 21.57 0 87 PMON72494 ZM_M26428 22 17.55 14.42 0 4 12.4 11.87 0.2114 29.95 26.29 1.00E−04 PMON72494 ZM_M26428 46 15.57 10.67 0 12 11.310.11 0.0033 29 26.86 20.78 0 PMON72494 ZM_M26428 23 14.1 11.43 0 13 97.98 0.0704 19 23.1 19.4 8.00E−04 PMON72494 ZM_M26428 −6 10.7 11.430.2402 9 8.71 7.98 0.1938 0 19.41 19.4 0.9925 PMON72494 ZM_M26428 311.02 10.67 0.5208 9 11.07 10.11 0.0163 6 22.09 20.78 0.1209 PMON72494ZM_M49327 8 12.13 11.23 0.2163 5 10.44 9.93 0.271 7 22.57 21.16 0.2103PMON72494 ZM_M49327 17 11.22 9.61 0.0189 4 8.28 7.93 0.5332 11 19.5117.53 0.0853 PMON72494 ZM_M49327 22 14.04 11.54 4.00E−04 21 9.73 8.060.0039 21 23.77 19.59 1.00E−04 PMON72494 ZM_M49328 4 11.7 11.23 0.511211 11.03 9.93 0.0196 7 22.74 21.16 0.1618 PMON72494 ZM_M49328 28 12.319.61 1.00E−04 17 9.27 7.93 0.0206 23 21.58 17.53 6.00E−04 PMON72494ZM_M49328 27 14.61 11.54 0 37 11.07 8.06 0 31 25.68 19.59 0 PMON72494ZM_M60546 −2 12.67 12.95 0.7032 6 9.48 8.95 0.4795 1 22.15 21.91 0.847889 PMON73765 ZM_M35084 10 10.56 9.61 0.1621 −2 7.8 7.93 0.8286 5 18.3617.53 0.4667 PMON73765 ZM_M35084 30 14.51 11.2 1.00E−04 27 9.25 7.270.0015 29 23.76 18.46 0 PMON73765 ZM_M54013 42 13.61 9.61 0 13 8.96 7.930.0717 29 22.57 17.53 0 PMON73765 ZM_M54013 32 14.78 11.2 0 49 10.827.27 0 39 25.6 18.46 0 PMON73765 ZM_M54016 33 12.82 9.61 0 7 8.51 7.930.3051 22 21.33 17.53 0.0013 PMON73765 ZM_M54016 34 14.98 11.2 0 3910.09 7.27 0 36 25.07 18.46 0 91 PMON73816 ZM_M37183 21 12.1 9.96 0.037814 10.65 9.35 0.0587 18 22.75 19.31 0.0348 PMON73816 ZM_M37183 33 11.58.66 0 21 9.82 8.09 0 27 21.32 16.75 0 PMON73816 ZM_M37188 18 11.78 9.960.076 21 11.3 9.35 0.0051 20 23.08 19.31 0.021 PMON73816 ZM_M37188 2410.71 8.66 0 16 9.41 8.09 3.00E−04 20 20.11 16.75 0 PMON73816 ZM_M3719730 12.93 9.96 0.0044 6 9.88 9.35 0.4306 18 22.82 19.31 0.0313 PMON73816ZM_M37197 30 11.26 8.66 0 13 9.11 8.09 0.0047 22 20.37 16.75 0 90PMON73829 ZM_M37805 29 9.46 7.32 1.00E−04 13 6.58 5.8 0.0171 22 16.0413.12 1.00E−04 PMON73829 ZM_M37805 18 11.78 9.96 0.076 15 10.74 9.350.0436 17 22.52 19.31 0.0484 PMON73829 ZM_M37815 30 12.92 9.96 0.0046 1310.57 9.35 0.0756 22 23.49 19.31 0.0109 PMON73829 ZM_M37815 11 8.14 7.320.1117 13 6.54 5.8 0.0225 12 14.68 13.12 0.0241 PMON73829 ZM_M38768 1311.26 9.96 0.201 −1 9.25 9.35 0.8842 6 20.51 19.31 0.4543 PMON73829ZM_M38768 −2 7.2 7.32 0.8084 2 5.93 5.8 0.6854 0 13.13 13.12 0.9914PMON73829 ZM_M38797 −39 4.49 7.32 0 −19 4.68 5.8 8.00E−04 −30 9.16 13.120 PMON73829 ZM_M38797 −11 8.83 9.96 0.2685 0 9.36 9.35 0.9827 −6 18.219.31 0.4895 PMON73829 ZM_M38798 −62 3.75 9.96 0 −35 6.07 9.35 0 −499.82 19.31 0 PMON73829 ZM_M38798 −50 3.67 7.32 0 −41 3.41 5.8 0 −46 7.0813.12 0 PMON73829 ZM_M39692 3 7.54 7.32 0.6671 −3 5.62 5.8 0.5857 013.16 13.12 0.9475 PMON73829 ZM_M39692 17 11.69 9.96 0.0919 3 9.59 9.350.7181 10 21.28 19.31 0.2211 92 PMON75305 ZM_M35696 26 14.78 11.77 0 1811.74 9.97 3.00E−04 22 26.52 21.74 0 PMON75305 ZM_M35696 33 11.51 8.66 015 9.33 8.09 7.00E−04 24 20.84 16.75 0 PMON75305 ZM_M36703 27 14.9411.77 0 13 11.25 9.97 0.007 20 26.19 21.74 0 PMON75305 ZM_M36703 4012.15 8.66 0 22 9.84 8.09 0 31 21.99 16.75 0 PMON75305 ZM_M36711 2614.88 11.77 0 9 10.91 9.97 0.0455 19 25.78 21.74 2.00E−04 PMON75305ZM_M36711 35 11.68 8.66 0 16 9.38 8.09 4.00E−04 26 21.06 16.75 0 93PMON75306 ZM_M35601 29 11.19 8.66 0 33 10.76 8.09 0 31 21.94 16.75 0PMON75306 ZM_M35601 11 13.05 11.77 0.0507 12 11.2 9.97 0.0097 11 24.2421.74 0.0159 PMON75306 ZM_M35604 24 14.64 11.77 0 16 11.57 9.97 9.00E−0421 26.21 21.74 0 PMON75306 ZM_M35604 42 12.29 8.66 0 35 10.92 8.09 0 3923.21 16.75 0 PMON75306 ZM_M35605 47 12.72 8.66 0 30 10.49 8.09 0 3923.2 16.75 0 PMON75306 ZM_M35605 18 13.92 11.77 0.0013 22 12.12 9.97 020 26.04 21.74 1.00E−04 94 PMON75309 ZM_M35865 21 10.45 8.66 0 3 8.38.09 0.5545 12 18.75 16.75 0.0017 PMON75309 ZM_M35865 22 11.75 9.660.0038 17 10.68 9.1 0.0064 20 22.43 18.76 0.0031 PMON75309 ZM_M35878 2310.6 8.66 0 26 10.17 8.09 0 24 20.78 16.75 0 PMON75309 ZM_M35878 1811.38 9.66 0.0163 13 10.3 9.1 0.0362 16 21.68 18.76 0.017 PMON75309ZM_M36160 19 11.51 9.66 0.0099 19 10.79 9.1 0.0037 19 22.31 18.76 0.0041PMON75309 ZM_M36160 32 11.41 8.66 0 19 9.6 8.09 0 25 21.01 16.75 0 95PMON75312 ZM_M35649 22 14.37 11.77 1.00E−04 12 11.18 9.97 0.0107 1825.55 21.74 3.00E−04 PMON75312 ZM_M35649 28 11.06 8.66 0 13 9.15 8.090.0034 21 20.21 16.75 0 PMON75312 ZM_M37099 9 9.46 8.66 0.0458 13 9.118.09 0.0049 11 18.57 16.75 0.0042 PMON75312 ZM_M37099 23 14.42 11.771.00E−04 10 10.97 9.97 0.0343 17 25.39 21.74 6.00E−04 PMON75312ZM_M37100 37 11.9 8.66 0 22 9.83 8.09 0 30 21.73 16.75 0 PMON75312ZM_M37100 9 12.85 11.77 0.0979 5 10.45 9.97 0.3064 7 23.29 21.74 0.1298101 PMON75515 ZM_M43539 26 12.88 10.19 0 13 10.12 8.98 0.0097 20 2319.17 0 PMON75515 ZM_M43546 −3 9.87 10.19 0.5762 −5 8.55 8.98 0.3141 −418.43 19.17 0.3786 PMON75515 ZM_M50136 16 10.41 8.98 0.0441 14 7.42 6.510.2064 15 17.84 15.48 0.085 PMON75515 ZM_M50136 24 13.2 10.68 0.0015 259.27 7.42 0.0053 24 22.47 18.1 4.00E−04 PMON75515 ZM_M50142 25 11.258.98 0.0018 17 7.61 6.51 0.1294 22 18.87 15.48 0.0145 PMON75515ZM_M50142 31 13.94 10.68 1.00E−04 35 10 7.42 1.00E−04 32 23.94 18.1 0105 PMON75524 ZM_M47998 17 11.23 9.61 0.0452 35 9.69 7.17 0.0012 2520.91 16.79 0.0043 PMON75524 ZM_M47998 15 13.3 11.54 0.0101 38 11.158.06 0 25 24.45 19.59 0 PMON75524 ZM_M48003 4 9.99 9.61 0.6366 9 7.787.17 0.4187 6 17.77 16.79 0.4837 PMON75524 ZM_M48003 28 14.77 11.54 0 159.22 8.06 0.0414 22 24 19.59 1.00E−04 PMON75524 ZM_M48004 19 11.44 9.610.0245 29 9.24 7.17 0.007 23 20.68 16.79 0.0069 PMON75524 ZM_M48004 512.11 11.54 0.3919 1 8.17 8.06 0.8374 4 20.28 19.59 0.5062 PMON75524ZM_M48005 18 11.37 9.61 0.0303 19 8.57 7.17 0.0654 19 19.93 16.79 0.0276PMON75524 ZM_M48005 33 15.38 11.54 0 29 10.4 8.06 1.00E−04 32 25.7819.59 0 PMON75524 ZM_M48007 20 11.51 9.61 0.0195 7 7.66 7.17 0.5152 1419.17 16.79 0.0927 PMON75524 ZM_M48007 28 14.78 11.54 0 46 11.78 8.06 036 26.55 19.59 0 PMON75524 ZM_M48010 22 11.77 9.61 0.0083 12 8.05 7.170.2443 18 19.81 16.79 0.0339 PMON75524 ZM_M48010 18 13.62 11.54 0.002625 10.08 8.06 6.00E−04 21 23.7 19.59 2.00E−04 107 PMON75533 ZM_M47453 5514.93 9.61 0 54 11.03 7.17 0 55 25.96 16.79 0 PMON75533 ZM_M47453 3914.99 10.8 0 44 10.24 7.12 0 41 25.23 17.92 0 PMON75533 ZM_M47460 1511.03 9.61 0.0782 5 7.53 7.17 0.63 11 18.56 16.79 0.208 PMON75533ZM_M47460 36 14.65 10.8 0 21 8.6 7.12 0.0037 30 23.25 17.92 0 PMON75533ZM_M49275 23 11.82 9.61 0.0069 20 8.58 7.17 0.0636 22 20.4 16.79 0.0119PMON75533 ZM_M49275 30 14.09 10.8 0 21 8.65 7.12 0.0028 27 22.74 17.92 0PMON75533 ZM_M49278 14 10.96 9.61 0.093 7 7.68 7.17 0.4982 11 18.6416.79 0.1885 PMON75533 ZM_M49278 18 12.79 10.8 0.0014 13 8.01 7.120.0757 16 20.8 17.92 0.0023 114 PMON75980 ZM_M53387 17 13.08 11.230.0122 11 10.99 9.93 0.0247 14 24.08 21.16 0.0109 PMON75980 ZM_M53389 1312.69 11.23 0.0463 9 10.85 9.93 0.0503 11 23.54 21.16 0.0363 PMON75980ZM_M53390 5 11.8 11.23 0.4269 4 10.33 9.93 0.3908 5 22.13 21.16 0.3859PMON75980 ZM_M53392 20 13.42 11.23 0.0033 13 11.19 9.93 0.0079 16 24.6221.16 0.0028 PMON75980 ZM_M53396 14 12.75 11.23 0.0383 4 10.38 9.930.338 9 23.12 21.16 0.0831 PMON75980 ZM_M53397 6 11.92 11.23 0.3398 −49.59 9.93 0.455 2 21.51 21.16 0.7576 PMON75980 ZM_M53398 4 11.66 11.230.5533 3 10.27 9.93 0.4659 4 21.93 21.16 0.4944 113 PMON78232 ZM_M55911−3 12.1 12.44 0.652 12 9.85 8.82 0.1004 3 21.94 21.27 0.5616 PMON78232ZM_M55911 −5 13.18 13.83 0.3591 2 9.43 9.27 0.8057 −2 22.61 23.09 0.6774PMON78232 ZM_M56069 14 14.13 12.44 0.031 7 9.44 8.82 0.3213 11 23.5621.27 0.0511 PMON78232 ZM_M56069 11 15.39 13.83 0.0296 12 10.38 9.270.0932 12 25.77 23.09 0.0237 PMON78232 ZM_M56206 −14 10.75 12.44 0.0307−9 8 8.82 0.1837 −12 18.75 21.27 0.0333 PMON78232 ZM_M56206 1 14.0313.83 0.7776 5 9.73 9.27 0.4808 3 23.76 23.09 0.5663 PMON78232 ZM_M5642812 13.9 12.44 0.0606 11 9.83 8.82 0.1065 12 23.73 21.27 0.0367 PMON78232ZM_M56428 13 15.55 13.83 0.0164 18 10.91 9.27 0.0143 15 26.46 23.090.0048 106 PMON79163 ZM_M45011 16 11.88 10.25 0.0215 7 8.54 8 0.4508 1220.42 18.26 0.0941 PMON79163 ZM_M45011 20 12.98 10.8 0.0017 23 8.74 7.120.0046 21 21.71 17.92 4.00E−04 PMON79163 ZM_M48217 16 11.89 10.25 0.021318 9.42 8 0.0487 17 21.3 18.26 0.0197 PMON79163 ZM_M48217 28 13.81 10.80 20 8.51 7.12 0.0062 24 22.32 17.92 0 98 PMON79174 ZM_M47171 13 11.5810.25 0.0602 20 9.61 8 0.0259 16 21.18 18.26 0.0247 PMON79174 ZM_M4717128 13.84 10.8 0 24 8.82 7.12 0.001 26 22.65 17.92 0 PMON79174 ZM_M4794118 12.09 10.25 0.0101 6 8.48 8 0.4971 13 20.57 18.26 0.0734 PMON79174ZM_M47941 25 13.53 10.8 0 16 8.24 7.12 0.026 21 21.77 17.92 1.00E−04 99PMON79413 ZM_M48525 44 13.83 9.61 0 30 9.34 7.17 0.0049 38 23.17 16.79 0PMON79413 ZM_M48525 26 13.66 10.8 0 32 9.41 7.12 0 29 23.07 17.92 0PMON79413 ZM_M50333 25 12.05 9.61 0.0031 25 8.95 7.17 0.0197 25 21 16.790.0036 PMON79413 ZM_M50333 27 13.75 10.8 0 34 9.55 7.12 0 30 23.3 17.920 PMON79413 ZM_M53171 18 11.34 9.61 0.0331 27 9.13 7.17 0.0107 22 20.4616.79 0.0106 PMON79413 ZM_M53171 21 13.04 10.8 3.00E−04 37 9.78 7.12 027 22.82 17.92 0 112 PMON79447 ZM_M53825 16 12.45 10.71 0.0079 17 9.127.83 0.0281 16 21.57 18.53 0.0077 PMON79447 ZM_M53825 30 14.57 11.21.00E−04 34 9.75 7.27 1.00E−04 32 24.32 18.46 0 PMON79447 ZM_M53826 1111.87 10.71 0.0705 0 7.84 7.83 0.9839 6 19.71 18.53 0.2903 PMON79447ZM_M53826 34 15 11.2 0 42 10.31 7.27 0 37 25.32 18.46 0 PMON79447ZM_M53835 6 11.31 10.71 0.342 −5 7.42 7.83 0.4779 1 18.73 18.53 0.8568PMON79447 ZM_M53835 32 14.83 11.2 0 47 10.66 7.27 0 38 25.49 18.46 0

4. Cold Field Efficacy Trial

This example sets forth a cold field efficacy trial to identify geneconstructs that confer enhanced cold vigor at germination and earlyseedling growth under early spring planting field conditions inconventional-till and simulated no-till environments. Seeds are plantedinto the ground around two weeks before local farmers are beginning toplant corn so that a significant cold stress is exerted onto the crop,named as cold treatment. Seeds also are planted under local optimalplanting conditions such that the crop has little or no exposure to coldcondition, named as normal treatment. The cold field efficacy trials arecarried out in five locations, including Glyndon Minn., Mason Mich.,Monmouth Ill., Dayton Iowa, Mystic Conn. At each location, seeds areplanted under both cold and normal conditions with 3 repetitions pertreatment, 20 kernels per row and single row per plot. Seeds are planted1.5 to 2 inch deep into soil to avoid muddy conditions. Two temperaturemonitors are set up at each location to monitor both air and soiltemperature daily.

Seed emergence is defined as the point when the growing shoot breaks thesoil surface. The number of emerged seedling in each plot is countedeveryday from the day the earliest plot begins to emerge until nosignificant changes in emergence occur. In addition, for each plantingdate, the latest date when emergence is 0 in all plots is also recorded.Seedling vigor is also rated at V3-V4 stage before the average of cornplant height reaches 10 inches, with 1=excellent early growth, 5=Averagegrowth and 9=poor growth. Days to 50% emergence, maximum percentemergence and seedling vigor are calculated using SAS software for thedata within each location or across all locations.

The following table lists the data that were collected and analyzedbased on the procedure illustrated above. The analyzed data across alllocations only include those from Glyndon Minn., Mason Mich., and MysticConn.

TABLE 19 Days to 50% Emergence Across Black Dirt Trts Warm TrtsMason_Trt 2 Glyndon_Trt 2 Mystic_Trt 2 PEP SEQ ID P P P P P constructEvent1 Delta value Delta value Delta value Delta value Delta value  88ZM_M31146 1.46 0.106 0.04 0.979 0.51 0.755 2.17 0.079 0.99 0.551pMON68399 ZM_M31147 1.29 0.153 0.34 0.81 0.59 0.721 1.47 0.234 1.640.322 ZM_M31524 −0.41 0.649 0.23 0.873 −2.09 0.205 −0.13 0.919 0.690.676 ZM_M32356 −0.21 0.814 0.33 0.815 −1.18 0.472 −1.59 0.197 3.520.034 Construct 0.53 0.302 0.24 0.772 −0.54 0.563 0.48 0.495 1.71 0.071 90 ZM_M37805 0.95 0.293 −0.04 0.977 −0.4 0.808 2.28 0.065 −0.35 0.831pMON73829 ZM_M37815 −1.24 0.169 0.06 0.965 −0.84 0.611 −1.4 0.258 −1.350.417 ZM_M38768 2.79 0.002 0.7 0.621 0.64 0.696 2.11 0.087 6.3 0   Construct 0.83 0.145 0.24 0.788 −0.2 0.849 1 0.2 1.53 0.144  92ZM_M35696 1.75 0.053 0.14 0.922 −1.93 0.24 4.17 0.001 0.61 0.715pMON75305 ZM_M36703 −0.47 0.603 0.4 0.777 −2.34 0.155 −0.83 0.502 2.120.202 ZM_M36711 −0.92 0.31 0.32 0.823 −1.23 0.454 −1.5 0.223 0.57 0.731Construct 0.12 0.832 0.29 0.749 −1.84 0.078 0.61 0.432 1.1 0.295  93ZM_M35601 −0.53 0.56 −0.36 0.803 −0.25 0.877 −0.22 0.861 −1.42 0.392pMON75306 ZM_M35604 −0.92 0.309 0.45 0.752 0.1 0.951 −1.89 0.125 −0 1   ZM_M35605 1.46 0.105 −0.08 0.958 −0.74 0.654 2.89 0.019 0.82 0.623Construct 0.01 0.992 0.01 0.994 −0.3 0.776 0.26 0.738 −0.2 0.847  94ZM_M35865 −0.31 0.735 −0.27 0.849 −2.91 0.078 0.25 0.84 1.18 0.475pMON75309 ZM_M35878 −0.1 0.916 0.33 0.817 0.3 0.858 −0.48 0.698 0.280.867 ZM_M36160 −0.58 0.519 −0.46 0.748 −1.84 0.264 −0.65 0.597 0.810.625 Construct −0.33 0.566 −0.13 0.882 −1.48 0.155 −0.29 0.707 0.760.47  107 ZM_M49275 −3.72 0.001 2.39 0.343 −5.47 0.004 −5.14 0 X XpMON75533 ZM_M49278 −2.37 0.042 2.08 0.409 −7.87 0 −1.9 0.185 X XConstruct −3.04 0.001 2.24 0.241 −6.67 0 −3.52 0.003 X X 119 ZM_M536411.25 0.166 0.04 0.978 2.88 0.081 −0.31 0.804 2.74 0.099 pMON78235ZM_M53994 −0.56 0.536 −0.13 0.926 −1.04 0.526 0.06 0.962 −1.31 0.429ZM_M53997 −0.8 0.376 0.11 0.937 0.38 0.816 −1.82 0.139 0.07 0.968Construct −0.04 0.95 0.01 0.994 0.74 0.478 −0.69 0.376 0.5 0.635 104ZM_M45248 −1.52 0.211 2.65 0.294 −4.77 0.013 −2.01 0.188 X X pMON78936ZM_M45274 −3.87 0.001 2.58 0.307 −5 0.009 −5.59 0 X X Construct −2.690.004 2.61 0.171 −4.89 0.002 −3.8 0.002 X X 110 ZM_M50823 −2 0.057 −0.170.921 −5.85 0.002 −2.51 0.08 2.87 0.136 pMON79425 ZM_M50856 0.01 0.993−0.6 0.714 −5.19 0.007 −0.31 0.839 6.23 0.001 ZM_M51300 −1.91 0.068−0.02 0.989 −4.43 0.021 −2.42 0.091 1.61 0.402 ZM_M51302 −3.5 0.001−0.21 0.899 −6.08 0.002 −5.38 0 2.85 0.139 ZM_M51313 −4.06 0 −0.12 0.94−4.16 0.03 −5.38 0 −1.31 0.496 ZM_M51608 −2.84 0.007 −0.27 0.87 −3.740.051 −4.88 0.001 2.15 0.265 ZM_M51623 −2.09 0.047 −0.15 0.926 −5.140.007 −3.15 0.028 3.09 0.11  Construct −2.34 0.001 −0.22 0.838 −4.94 0−3.43 0 2.5 0.048 116 ZM_M53939 −2.66 0.022 2.55 0.313 −3.3 0.085 −4.630.001 X X pMON79697 ZM_M54371 −1.02 0.378 2.71 0.282 −3.56 0.063 −2.040.154 X X ZM_M54374 −2.79 0.016 2.67 0.29 −4.36 0.023 −4.3 0.003 X XConstruct −2.16 0.01 2.64 0.11 −3.74 0.009 −3.66 0.001 X X 111 ZM_M51598−2.19 0.071 2.23 0.376 −4.51 0.019 −3.25 0.033 X X pMON79718 ZM_M52937−1.8 0.138 3.07 0.224 −5.32 0.006 −2.14 0.162 X X Construct −2 0.0372.65 0.165 −4.92 0.002 −2.69 0.028 X X 120 ZM_M53455 0.14 0.873 0.290.838 3.04 0.065 −1.71 0.166 0.95 0.565 pMON80452 ZM_M53456 −0.56 0.532−0.51 0.719 0.97 0.555 −1.18 0.337 −0.86 0.602 ZM_M53694 0.88 0.332 0.250.859 2.06 0.211 1.04 0.401 −0.62 0.706 ZM_M53695 1.47 0.104 0 0.9983.07 0.062 0.22 0.857 2.37 0.154 ZM_M53696 0.95 0.295 −0.2 0.888 0.460.78 0.74 0.55 1.85 0.265 Construct 0.57 0.23 −0.03 0.965 1.92 0.028−0.18 0.783 0.74 0.402 118 ZM_M53218 −1.55 0.087 −0.02 0.988 −3.54 0.032−2.09 0.09 1.55 0.351 pMON80461 ZM_M53235 −1.42 0.117 0.34 0.808 −0.50.761 −1.86 0.131 −1.44 0.386 ZM_M53848 −0.36 0.69 −0.02 0.988 −1.11 0.5−0.6 0.624 0.88 0.595 ZM_M54282 −0.98 0.279 0.16 0.909 −3.97 0.016 0.320.796 −0.58 0.727 ZM_M54284 −1.06 0.24 0.05 0.972 −0.35 0.832 −1.210.328 −1.49 0.37  Construct −1.07 0.025 0.1 0.891 −1.89 0.03 −1.09 0.095−0.21 0.806 Maximum Percent Emergence Across Black Dirt Trts Warm TrtsMason_Trt 2 Glyndon_Trt 2 Mystic_Trt 2 PEP SEQ ID P P P P P constructEvent1 Delta value Delta value Delta value Delta value Delta value  88ZM_M31146 −2.7 0.428 1.42 0.601 0.19 0.97 −7.53 0.125  4.07 0.503pMON68399 ZM_M31147 −6.31 0.064 −5.8 0.033 −6.48 0.184 −9.75 0.047  0.740.903 ZM_M31524 −2.7 0.428 −1.91 0.481 5.19 0.288 −4.2 0.393 −7.59 0.212ZM_M32356 3.55 0.297 −1.91 0.481 8.52 0.081 5.8 0.237 −5.93 0.33 Construct −2.04 0.293 −2.05 0.185 1.85 0.505 −3.92 0.161 −2.18 0.53   90ZM_M37805 −4.18 0.22 5.83 0.032 1.67 0.733 −9.01 0.067 −0.37 0.951pMON73829 ZM_M37815 4.71 0.167 −3.62 0.183 8.33 0.088 2.1 0.669 6.30.301 ZM_M38768 −6.27 0.066 −2.51 0.356 −1.67 0.733 −5.68 0.247 12.040.048 Construct −1.91 0.374 −0.1 0.954 2.78 0.368 −4.2 0.177 −2.04 0.596 92 ZM_M35696 −5.02 0.141 2.49 0.359 10 0.041 12.35 0.012 −5.37 0.377pMON75305 ZM_M36703 0.4 0.906 −1.95 0.473 6.67 0.172 0.99 0.841 −7.040.248 ZM_M36711 3.6 0.291 1.38 0.611 1.67 0.733 6.54 0.183 −0.37 0.951Construct −0.34 0.875 0.64 0.709 6.11 0.048 −1.6 0.605 −4.26 0.268  93ZM_M35601 −2.52 0.46 3.6 0.185 1.67 0.733 −5.68 0.247 −0.37 0.951pMON75306 ZM_M35604 3.04 0.372 −3.06 0.26 5 0.305 5.43 0.269 −3.7  0.543ZM_M35605 −3.49 0.306 −1.4 0.607 8.33 0.088 10.12 0.039 −2.04 0.738Construct −0.99 0.647 −0.28 0.869 5 0.105 −3.46 0.266 −2.04 0.596  94ZM_M35865 −2.1 0.538 −3.06 0.315 1.67 0.733 −2.35 0.633 −5.37 0.377pMON75309 ZM_M35878 −0.99 0.772 1.38 0.611 −0 1 −0.12 0.98 −3.7  0.543ZM_M36160 0.82 0.81 1.38 0.611 8.33 0.088 0.99 0.841 −7.04 0.248Construct −0.76 0.726 −0.1 0.955 3.33 0.28 −0.49 0.874 −5.37 0.163 107ZM_M49275 10.25 0.019 5.28 0.274 17.5 0.002 15 0.009 X X pMON75533ZM_M49278 4.88 0.265 −1.39 0.773 19.17 0.001 6.11 0.284 X X Construct7.56 0.03 1.94 0.594 18.33 0 10.56 0.024 X X 119 ZM_M53641 −1.27 0.711.38 0.611 5 0.305 0.99 0.841 12.04 0.048 pMON78235 ZM_M53994 1.65 0.628−1.4 0.607 3.33 0.494 0.99 0.841 1.3 0.831 ZM_M53997 5.26 0.122 3.050.262 1.67 0.733 9.88 0.044 −0.37 0.951 Construct 1.86 0.382 1.01 0.5573.33 0.28 3.95 0.203 −3.7  0.336 104 ZM_M45248 −3.09 0.481 1.94 0.68710.83 0.056 −1.67 0.77 X X pMON78936 ZM_M45274 9.88 0.024 6.94 0.1514.17 0.013 16.11 0.005 X X Construct 3.39 0.331 4.44 0.223 12.5 0.0077.22 0.121 X X 110 ZM_M50823 4.65 0.24 −0.83 0.792 10.83 0.056 7.220.206 −6.67 0.346 pMON79425 ZM_M50856 −6.88 0.082 −0.83 0.792 5.83 0.304−8.33 0.144 16.67 0.019 ZM_M51300 3.54 0.371 −0.83 0.792 4.17 0.462 8.330.144 −6.67 0.346 ZM_M51302 12.85 0.001 0.83 0.792 14.17 0.013 19.440.001 −1.67 0.814 ZM_M51313 9.51 0.016 −0.83 0.792 15.83 0.005 12.780.025 −3.33 0.637 ZM_M51608 5.49 0.166 0.83 0.792 10.83 0.056 7.22 0.206−3.33 0.637 ZM_M51623 1.6 0.687 3.06 0.333 17.5 0.002 2.78 0.626 16.670.019 Construct 4.39 0.09 0.2 0.923 11.31 0.002 7.06 0.059 −7.86 0.09 116 ZM_M53939 6.36 0.147 1.94 0.687 12.5 0.028 11.67 0.041 X X pMON79697ZM_M54371 0.06 0.989 3.61 0.454 2.5 0.659 7.22 0.206 X X ZM_M54374 10.060.022 −1.39 0.773 12.5 0.028 17.22 0.003 X X Construct 5.49 0.081 1.390.66 9.17 0.03 12.04 0.005 X X 111 ZM_M51598 4.13 0.345 −1.39 0.77319.17 0.001 5 0.381 X X pMON79718 ZM_M52937 −1.42 0.745 6.94 0.15 15.830.005 −1.67 0.77 X X Construct 1.36 0.698 2.78 0.446 17.5 0 1.67 0.72 XX 120 ZM_M53455 1.65 0.628 −1.95 0.473 −3.33 0.494 7.65 0.119 −5.370.377 pMON80452 ZM_M53456 3.04 0.372 0.27 0.921 −5 0.305 8.77 0.074−0.37 0.951 ZM_M53694 −0.15 0.964 0.83 0.761 −1.67 0.733 −0.12 0.98 1.30.831 ZM_M53695 −3.9 0.252 1.38 0.611 1.67 0.733 −3.46 0.481 10.37 0.089ZM_M53696 0.96 0.779 2.49 0.359 6.67 0.172 2.1 0.669 −7.04 0.248Construct 0.32 0.86 0.6 0.675 −0.33 0.897 2.99 0.25 −4.37 0.175 118ZM_M53218 3.46 0.31 −0.84 0.757 8.33 0.088 8.77 0.074 12.04 0.048pMON80461 ZM_M53235 3.6 0.291 0.83 0.761 −3.33 0.494 9.88 0.044 −2.040.738 ZM_M53848 4.98 0.143 3.05 0.262 6.67 0.172 7.65 0.119 −2.04 0.738ZM_M54282 −0.57 0.867 −3.62 0.183 6.67 0.172 −3.46 0.481 −2.04 0.738ZM_M54284 4.98 0.143 −1.19 0.679 10 0.041 0.99 0.841  7.96 0.191Construct 3.29 0.068 −0.35 0.807 5.67 0.028 4.77 0.067 −2.04 0.527E. Screens for Transgenic Plant Seeds with Increased Protein and/or OilLevels

This example sets forth a high-throughput selection for identifyingplant seeds with improvement in seed composition using the Infratec 1200series Grain Analyzer, which is a near-infrared transmittancespectrometer used to determine the composition of a bulk seed sample.Near infrared analysis is a non-destructive, high-throughput method thatcan analyze multiple traits in a single sample scan. An NIR calibrationfor the analytes of interest is used to predict the values of an unknownsample. The NIR spectrum is obtained for the sample and compared to thecalibration using a complex chemometric software package that provides apredicted values as well as information on how well the sample fits inthe calibration.

Infratec Model 1221, 1225, or 1227 with transport module by Foss NorthAmerica is used with cuvette, item #1000-4033, Foss North America or forsmall samples with small cell cuvette, Foss standard cuvette modified byLeon Girard Co. Corn and soy check samples of varying compositionmaintained in check cell cuvettes are supplied by Leon Girard Co. NITcollection software is provided by Maximum Consulting Inc. Software.Calculations are performed automatically by the software. Seed samplesare received in packets or containers with barcode labels from thecustomer. The seed is poured into the cuvettes and analyzed as received.

TABLE 20 Typical sample(s): Whole grain corn and soybean seedsAnalytical time Less than 0.75 min per sample to run method: Totalelapsed 1.5 minute per sample time per run: Typical and minimum Corntypical: 50 cc; minimum 30 cc sample size: Soybean typical: 50 cc;minimum 5 cc Typical analytical Determined in part by the specificrange: calibration. Corn - moisture 5-15%, oil 5-20%, protein 5-30%,starch 50-75%, and density 1.0-1.3%. Soybean - moisture 5-15%, oil15-25%, and protein 35-50%.

TABLE 21 Kernel Protein Content of Transgenic plant seeds in MidwestHybrid Trials in 2003, 2004, and 2005. Hybrid 2003 Hybrid 2004 Hybrid2005 PEP Mean Mean Mean SEQ Trans- Mean % Pval- Trans- Mean % Pva-Trans- Mean % Pval- ID Construct Event genic Control^(a) Change ue genicControl^(a) Change lue genic Control^(b) Change ue 84 PMON69462ZM_M17475 9.2 8.7 6.9 0.00 8.8 8.1  8.8 0.00 9.5 9.0 6.4 0.00 PMON69462ZM_M17512 9.4 8.7 8.0 0.00 8.9 8.1 10.3 0.00 9.6 9.0 6.8 0.00 PMON69462ZM_M19779 8.6 8.7 −1.1  0.37 8.0 8.1 −1.8 0.20 — — — — PMON69462ZM_M19792 8.9 8.7 2.3 0.17 8.1 8.1 −0.1 0.92 — — — — PMON69462 ZM_M197758.5 8.7 −2.3  0.17 8.0 8.1 −1.4 0.32 — — — — PMON69462 ZM_M19755 — — — —8.1 8.1  0.3 0.83 8.7 9.0 −2.5  0.09 PMON69462 ZM_M19263 — — — — 7.9 8.1−2.1 0.12 — —_(—) — — PMON69462 ZM_M19752 — — — — 8.1 8.1  0.0 0.97 — —— — 126 PMON83769 ZM_M75771 — — — — — — — — 9.6 9.1 5.9 0.00 PMON83769ZM_M73623 — — — — — — — — 9.1 9.1 0.2 0.92 PMON83769 ZM_M73624 — — — — —— — — 9.8 9.1 7.7 0.00 PMON83769 ZM_M74392 — — — — — — — — 9.6 9.1 5.00.00 PMON83769 ZM_M74394 — — — — — — — — 9.9 9.1 8.8 0.00 PMON83769ZM_M74395 — — — — — — — — 9.5 9.1 4.5 0.01 PMON83769 ZM_M75255 — — — — —— — — 9.8 9.1 8.0 0.00 PMON83769 ZM_M75260 — — — — — — — — 9.5 9.1 4.10.01 124 PMON80868 ZM_M59335 — — — — — — — — 9.2 9.0 2.1 0.24 PMON80868ZM_M59391 — — — — — — — — 9.3 9.0 3.0 0.10 PMON80868 ZM_M59764 — — — — —— — — 9.0 9.0 0.0 0.98 Kernel protein reported on a 100% dry matterbasis ^(a)Control for 2003 and 2004 was recurrent parent ^(b)Control for2005 trial was pollinator for pMON69462 and recurrent parent forpMON83769 and pMON80868

TABLE 22 Kernel Protein Content of Transgenic plant seeds in HawaiiInbred Trials^(a) PEP SEQ Mean Mean % ID Construct Event Year TransgenicControl^(b) Change Pvalue 84 PMON69462 ZM_M17475 2002 14.2 10.7 32.70.02 PMON69462 ZM_M17512 2002 12.6 11.8 6.8 0.10 PMON69462 ZM_M197792002 11.4 10.7 6.5 0.10 PMON69462 ZM_M19792 2002 12.5 11.6 7.8 0.10PMON69462 ZM_M19775 2002 12.9 11.9 8.4 0.10 PMON69462 ZM_M19755 200312.0 11.3 6.4 0.44 PMON69462 ZM_M19263 2003 10.8 11.0 −2.2 0.77PMON69462 ZM_M19752 2003 11.1 11.9 −7.0 0.23 PMON69462 ZM_M19270 200213.0 10.5 23.8 0.02 PMON69462 ZM_M19781 2002 12.4 10.3 20.4 0.02PMON69462 ZM_M19257 2003 12.7 11.4 11.4 0.30 126 PMON83769 ZM_M736242004 13.4 9.4 42.9 0.00 PMON83769 ZM_M74380 2004 11.9 11.7 1.4 0.88PMON83769 ZM_M74392 2004 10.7 12.0 −10.5 0.21 PMON83769 ZM_M74394 200411.8 10.7 10.5 0.05 PMON83769 ZM_M74395 2004 13.6 11.8 14.8 0.00PMON83769 ZM_M75255 2004 12.5 11.0 13.2 0.27 PMON83769 ZM_M75771 200412.3 12.5 −2.2 0.83 124 PMON80868 ZM_M59335 2004 13.3 12.1 10.4 0.07PMON80868 ZM_M59764 2004 12.8 11.5 10.8 0.27 PMON80868 ZM_M59765 200413.7 11.8 15.5 0.00 ^(a)Kernel protein reported on a 100% dry matterbasis ^(b)Control was negative isoline for each event

Example 6

This example illustrates the preparation of transgenic plant cellscontaining recombinant DNA (SEQ ID NO:82) expressing a maize phytochromeA protein (PHYA). A full-length cDNA encoding a corn PHYA protein wascloned from corn. The cDNA clone contained 3396 bp of nucleotidesencoding a 1131 amino acid PHYA protein with molecular weight at 125.2kD. Based on the cDNA sequences, primers were designed to clone agenomic DNA, illustrated in FIG. 1, from a maize inbred LH172 genomiclibrary. Recombinant DNA comprising a rice actin promoter operablylinked to the genomic DNA encoding the corn PHYA protein followed by aHsp17 terminator was inserted into transformation vector of pMON74916 asset forth in SEQ ID NO:10030. Corn plant cells were transformed withrecombinant DNA expressing PHA using pMON74916 and used to regenerate apopulation of transgenic plants. Transgenic plants were regenerated fromabout 100 events of transformed plant cells; plants from 90 of theevents with various expression levels were selected for pollination toproduce R1 and F1 seeds; and plants from 31 events were selected forscreening for an enhanced trait.

Seed Germination and Seedling Development

Five events were selected to be analyzed phenotypic effect on seedgermination and seedling development in the dark condition along withother transgenic material. 12 inbred seeds of each wild-type andtransgenic maize events were germinated in a wetted and rolledgermination paper in a complete dark growth chamber for 10 days. Thelength of mesocotyl, coleoptiles and root were measured for everyseedling. The transgenic maize seedlings were identified showing greatelongation growth of both mesocotyl and expanded coleoptiles impartedfrom recombinant DNA expressing PHYA protein as compared tonon-transgenic controls. Density Study

Transgenic plants were grown in fields at three densities: high densityat 42,000 plants per acre; medium density at 35,000 plants per acre; andlow density at 28,000 plants per acre. Plants from three plant cellevents expressing PHYA were selected for studying physiological andyield responses to different densities. The physiological data from thedensity trial YI130 is summarized in the Table 23 shown below. EventZM_S83483 under high planting density showed significant decrease inplant height, ear height, and internode length and had a significantincrease in chlorophyll content.

TABLE 23 Low Density YI130 JV 2004 High Density YI130 JV 2004 Event StemInternode Internode Photo ID Plant Height Ear Height Diameter LengthPlant Height Ear Height Length SPAD rate ZM_S83483 not Significant notSignificant not Significant increase not Significant NA significantDecrease significant Decrease significant Decrease significant IncreaseP = 0.727 P = 0.085 P = 0.9436 P = 0.0370 P = 0.5866 P = 0.0185 P =0.2412 P = 0.0762 ZM_S83897 not decrease not not Significant Significantnot not Significant Significant significant significant significantIncrease Decrease significant significant Decrease Decrease P = 0.8778 P= 0.1937 P = 0.2517 P = 0.0421 P = 0.0306 P = 0.6542 P = 0.5206 P =0.0153 ZM_S83907 Highly increase not Significant Highly SignificantSignificant not increase not not Significant significant IncreaseSignificant Increase Increase significant significant significantIncrease P = 0.2426 P = 0.0633 Increase P = 0.0016 P = 0.015 P = 0.89 P= 0.3208 P = 0.0021 P = 0.001

Kernel Trait Analysis

As shown in Table 24, events ZM_S83444 ZM_S83446, ZM_S83473, ZM_S83480,ZM_S83483, and ZM_S83907 show significant increases in single kernelweight. Event ZM_S83452 shows significant increases in single kernelweight and total kernel weight. The screening data show that plant cellswith stably-integrated, non-natural, recombinant DNA expressing aphytochrome A protein can be regenerated into plants exhibitingincreased yield as compared to control plants.

event Trait Mean_TRAN Mean_CON TRAN-CON % change Pvalue Result ZM_S83416Total kernel weight, g 151.3 140.21 11.09 8 0.1452 Non SignifincantTotal kernel number 876 830.22 45.78 6 0.3118 Non Signifincant Singelkernel weight, g 0.17 0.17 0.01 6 0.3551 Non Signifincant ZM_S83444Total kernel weight, g 147.14 144.65 2.49 2 0.753 Non Signifincant Totalkernel number 664.38 930.47 −266.1 −29 0 Highly Significant Singelkernel weight, g 0.25 0.16 0.09 56 0 Highly Significant ZM_S83446 Totalkernel weight, g 152.12 158.27 −6.15 −4 0.3931 Non Signifincant Totalkernel number 718.88 918.94 −200.07 −22 0 Highly Significant Singelkernel weight, g 0.2 0.17 0.03 18 0.0008 Highly Significant ZM_S83452Total kernel weight, g 166.94 140.21 26.72 19 0.0014 Highly SignificantTotal kernel number 888.89 830.22 58.67 7 0.2123 Non Signifincant Singelkernel weight, g 0.19 0.17 0.02 12 0.0045 Highly Significant ZM_S83473Total kernel weight, g 145.87 146.47 −0.6 −0 0.9451 Non SignifincantTotal kernel number 784.71 885.21 100.5 −11 0.0099 Highly SignificantSingel kernel weight, g 0.18 0.16 0.02 13 0.0618 Signifincant at 10%ZM_S83480 Total kernel weight, g 157.23 149.44 7.79 5 0.3769 NonSignifincant Total kernel number 856.67 924.28 −67.61 −7 0.0982Signifincant at 10% Singel kernel weight, g 0.18 0.16 0.02 13 0.0018Highly Significant ZM_S83483 Total kernel weight, g 164.86 158.27 6.6 40.3599 Non Signifincant Total kernel number 820.4 918.94 −98.54 −110.0165 Significant Singel kernel weight, g 0.19 0.17 0.02 12 0.0317Significant ZM_S83897 Total kernel weight, g 132.62 149.44 −16.83 −110.0617 Signifincant at 10% Total kernel number 743.5 924.28 −180.78 −200.0001 Highly Significant Singel kernel weight, g 0.18 0.16 0.02 130.0125 Significant ZM_S83907 Total kernel weight, g 146.23 146.47 −0.24−0 0.9807 Non Signifincant Total kernel number 733.44 833.41 −99.97 −120.0703 Signifincant at 10% Singel kernel weight, g 0.19 0.17 0.02 120.0792 Signifincant at 10% ZM_S83416 Total kernel weight, g 157.3 146.4710.83 7 0.2666 Non Signifincant Total kernel number 881.8 833.41 48.39 60.3558 Non Signifincant Singel kernel weight, g 0.18 0.17 0 0 0.6827 NonSignifincant

Example 7

This example illustrates the preparation of transgenic plant cellscontaining recombinant DNA (SEQ ID NO:77) expressing a soybean MADS boxtranscription factor protein and identified as G 1760.

The DNA encoding the soybean MADS box transcription factor was clonedfrom a soybean library and inserted into a recombinant DNA constructcomprising a CaMV 35S promoter operably linked to the DNA encoding thetranscription factor followed by a terminator. The recombinant DNAconstruct was inserted into a transformation vector plasmid to produceplasmid pMON74470, as set forth in SEQ ID NO: 10029 which was used forAgrobacterium-mediated transformation of soybean plant cells.

Soybean plant cells were transformed with recombinant DNA expressing theMADS box transcription factor using MON74470 and used to regenerate apopulation of transgenic plants. Transgenic soybean plants wereregenerated and selected for screening for an enhanced trait.

Transgenic soybean plants exhibited flowers with highly enlarged sepalsand a winding stem. The main stem exhibited reduced lateral branchingand increased raceme formation. Flowering time was decreased by about 2to 4 days as compared to control plants under short day (10 hr) and longday (14 hr) conditions. Transgenic plants also flowered by 5 weeks whenplaced under non-inductive 20 hr light; wild-type control plants did notflower under such conditions. Floral and pod abscission was greatlyreduced in the transgenic plants resulting in an increase in the numberof pods per plant. Wild type control plants produced on the order of 100pods, specific transgenic plants produced at least 125 pods per plantand plants regenerated from plant cells of one transgenic event producedgreater than 200 pods per plant. There was also a delay in maturityranging from one week exhibited by plants from single copy event A29204to a month exhibited by plants from a multi-copy event A28877. Over 95%of the pods on transgenic plants from event A29204 mature in a timeperiod; but only 50% of the pods on transgenic plants from event A28877mature in the same time period. Seeds from transgenic plants weresmaller than seed from control plants and greater in number than seedsfrom control plants, e.g. about 1800 more seed per pound. Transgenicplants were also shown to be have enhanced water use efficiency.

In testing soybeans for drought tolerance, 4.5″ pots were prepared withMetromix 200 and the pots were adjusted to the same weight. Pots weresaturated with water. R2 or R3 homozygous seeds were placed in the soilin the pots, 15 pots per event, 3 to 6 events per construct. Plants weregrown with a light intensity of 600 μEM⁻²S⁻¹; Temperature: 28° C.;Relative humidity (RH): 60%. A gene check with gene check strip (TraitRUR Lateral Flow 50 tests, from Strategic Diagnostics, Inc.) for thepresence of the CP4 gene was done on selected plants. Unwanted negativeplants were discarded. When plants reached the V1 stage. Pots weresaturated with water by thorough irrigation. A picture was taken of theplant in the water saturated pot. Excess water was drained and furtherwater was withheld until the pot water content of 50% and 10% of thewater capacity for well watered controls and drought treated plants,respectively (monitor the water content by measuring soil moisture orpot weight every 3-5 days). At approximately 10% of the saturated waterweight, the plants began to show the onset of the wilting phenotype.Limited-watering was continued every 1-2 days to maintain pot watercontent at 50 or 10%. The drought injury phenotype was determined fornext 14 days (see the table of measurements). Photograph of plants andphysiological assays were run on each at 14 days after the onset ofdrought treatment. Theses included, but were not limited to, plantheight, leaf relative water content, leaf water potential, chlorophyllcontent and chlorophyll fluorescence. Pot were saturated with nutrientsolution and resume regular watering schedule after 14 days.

TABLE 25 Measurement Protocol Agronomic measurements Emergence, earlyseason vigor, height (cm) Visual drought score Score of 1 to 4: 1.Healthy plants, no difference from control plants; 2. On sight ofwilting, leaves become wilt; 3. Wilted plants, still green andrecoverable; 4. Severely wilted, chlorotic and not recoverableDrought assay measurements as described in Table 25 taken on transgenicsoybean plants showed that transgenic soybean plants from transgenicplant cells of event GM 29204 exhibited enhanced water use efficiency.

R0 plants regenerated from one transgenic plant cell event (28877) of 41transgenic plant cells events produced a large number of pods per nodeand seeds/plant−531 R1 seeds per plant compared to an average of 150seeds per plant, i.e. increased yield.

Example 6 Consensus Sequence

This example illustrates the identification of consensus amino acidsequence for the proteins and homologs encoded by DNA that is used toprepare the transgenic seed and plants of this invention having enhancedagronomic traits.

ClustalW program was selected for multiple sequence alignments of theamino acid sequence of SEQ ID NO: 136 and its nine homologs, and SEQ IDNO: 151 and its 11 homologs. Three major factors affecting the sequencealignments dramatically are (1) protein weight matrices; (2) gap openpenalty; (3) gap extension penalty. Protein weight matrices availablefor ClustalW program include Blosum, Pam and Gonnet series. Thoseparameters with gap open penalty and gap extension penalty wereextensively tested. On the basis of the test results, Blosum weightmatrix, gap open penalty of 10 and gap extension penalty of 1 werechosen for multiple sequence alignment. FIG. 2 shows the sequences ofSEQ ID NO: 136, its homologs and the consensus sequence (SEQ ID NO:10031) at the end. FIG. 3 shows the sequences of SEQ ID NO: 151, itshomologs and the consensus sequence (SEQ ID NO: 10032) at the end. Thesymbols for consensus sequence are (1) uppercase letters for 100%identity in all positions of multiple sequence alignment output; (2)lowercase letters for >=70% identity; symbol; (3) “X” indicated <70%identity; (4) dashes “-” meaning that gaps were in >=70% sequences.

The consensus amino acid sequence can be used to identify DNAcorresponding to the full scope of this invention that is useful inproviding transgenic plants, for example corn and soybean plants withenhanced agronomic traits, for example improved nitrogen use efficiency,improved yield, improved water use efficiency and/or improved growthunder cold stress, due to the expression in the plants of DNA encoding aprotein with amino acid sequence identical to the consensus amino acidsequence.

Example 7 Identification of Amino Acid Domain by Pfam Analysis

The amino acid sequence of the expressed proteins that were shown to beassociated with an enhanced trait were analyzed for Pfam protein familyagainst the current Pfam collection of multiple sequence alignments andhidden Markov models using the HMMER software in the appended computerlisting. The Pfam protein families for the proteins of SEQ ID NO:84through 166 are shown in Table 26. The Hidden Markov model databases forthe identified patent families are also in the appended computer listingallowing identification of other homologous proteins and their cognateencoding DNA to enable the full breadth of the invention for a person ofordinary skill in the art. Certain proteins are identified by a singlePfam domain and others by multiple Pfam domains. For instance, theprotein with amino acids of SEQ ID NO: 91 is characterized by two Pfamdomains, i.e. SRF-TF and K-box; and, the protein with amino acids of SEQID NO:165 is characterized by six Pfam domains, i.e. GAF, Phytochrome,PAS, a repeated PAS, HisKA, and HATPase.

TABLE 26 NUC SEQ ID PEP SEQ ID Pfam domain name begin stop score E-value3 86 Pkinase 79 337 343  4.30E−100 5 88 FA_desaturase 99 319 206.26.60E−59 2 85 Ras 10 178 297.9 1.60E−86 1 84 Glyoxalase 27 171 130.15.40E−36 8 91 SRF-TF 9 59 121.4 2.30E−33 8 91 K-box 75 176 151.71.70E−42 7 90 K-box 4 104 145.6 1.20E−40 83 166 SRF-TF 9 59 99.21.10E−26 83 166 K-box 75 172 92.4 1.20E−24 82 165 GAF 219 404 105.61.30E−28 82 165 Phytochrome 415 595 407.6  1.60E−119 82 165 PAS 622 73888.9 1.40E−23 82 165 PAS 753 878 101.1 2.80E−27 82 165 HisKA 898 95727.6 4.00E−05 82 165 HATPase_c 1012 1124 66.9 5.80E−17 9 92 Homeobox 97158 68 2.80E−17 10 93 AP2 5 68 127.5 3.30E−35 11 94 GATA 196 231 71.32.70E−18 12 95 AT_hook 57 69 7.4 1.1   12 95 DUF296 84 208 183.64.30E−52 24 107 Synaptobrevin 128 215 137.6 2.90E−38 31 114Pyridoxal_deC 28 381 194.6 2.10E−55 36 119 Metallophos 63 258 1612.80E−45 21 104 Pkinase 12 267 346  5.40E−101 21 104 Pkinase_Tyr 12 26588.5 1.80E−23 21 104 NAF 310 369 98.6 1.60E−26 26 109 MtN3_slv 9 98 96.76.10E−26 26 109 MtN3_slv 132 218 116.8 5.70E−32 27 110 Lactamase_B 94252 125.1 1.80E−34 33 116 HSP20 53 157 159.9 5.80E−45 28 111 RTC 3 353275.2 1.10E−79 28 111 RTC_insert 184 300 120.8 3.40E−33 37 120 PDZ 200284 37.6 3.80E−08 37 120 Peptidase_S41 320 483 244.5 1.90E−70 35 118E2F_TDP 167 232 131 2.90E−36 41 124 Pkinase 63 341 199.5 7.00E−57 41 124Pkinase_Tyr 63 341 243 5.60E−70 43 126 zf-C2H2 72 94 25.6  0.00016 43126 zf-C2H2 149 171 20.5 0.0054 4 87 zf-C2H2 85 107 22.1 0.0018 17 100PRA1 10 161 181.8 1.50E−51 22 105 AAA 154 352 85 2.10E−22 14 97CBFD_NFYB_HMF 31 96 134.4 2.80E−37 34 117 Peptidase_C15 11 219 −72.23.50E−07 20 103 Pkinase 13 267 345.5  7.80E−101 20 103 Pkinase_Tyr 13265 75.2 1.80E−19 20 103 NAF 312 371 104.7 2.50E−28 32 115 HSF_DNA-bind49 225 212.2 1.00E−60 19 102 Pkinase 37 291 353.9  2.30E−103 19 102 RIO150 208 −88.1 0.0038 19 102 NAF 375 432 101.8 1.80E−27 40 123Aldo_ket_red 7 284 448.1  1.00E−131 42 125 FBPase 13 337 691.6 5.30E−205 6 89 SRF-TF 9 59 119.7 7.20E−33 18 101 DNA_photolyase 6 173163.3 5.70E−46 18 101 FAD_binding_7 205 476 425.8  5.50E−125 30 113Pkinase 41 327 326.6 3.80E−95 23 106 NIF 95 291 90.6 4.10E−24 15 98 Got130 130 237 3.60E−68 16 99 RRM_1 21 89 67.1 5.00E−17 29 112 Di19 13 206365.4  8.00E−107 25 108 CorA 90 467 408.2  1.00E−119 39 122 SPC25 12 190252.3 9.00E−73 44 127 Response_reg 18 139 151.1 2.60E−42 44 127 HisKA320 385 101.5 2.30E−27 44 127 HATPase_c 432 565 138.4 1.70E−38 44 127Response_reg 740 862 128 2.40E−35 44 127 Hpt 922 1013 63.4 6.60E−16 45128 Response_reg 18 139 151.1 2.60E−42 45 128 HisKA 320 385 101.52.30E−27 45 128 HATPase_c 432 565 138.4 1.70E−38 45 128 Response_reg 740862 128 2.40E−35 45 128 Hpt 922 1013 63.4 6.60E−16 46 129 NAM 9 135313.7 2.90E−91 47 130 Aminotran_1_2 183 576 55.7 1.40E−13 48 131Catalase 18 401 960.1  7.80E−286 49 132 BRO1 10 172 177.8 2.40E−50 69152 Got1 30 130 211.8 1.40E−60 70 153 Got1 30 130 174.9 1.80E−49 71 154Cystatin 36 124 87.6 3.40E−23 72 155 Cystatin 36 124 87.6 3.40E−23 73156 RRM_1 22 87 32.4 1.40E−06 74 157 Pkinase_Tyr 55 304 86.2 9.10E−23 74157 Pkinase 55 306 362  8.40E−106 75 158 SPX 1 167 88.9 1.30E−23 75 158zf-C3HC4 238 286 17 0.0024 76 159 Pkinase_Tyr 19 271 70.8 4.00E−18 76159 Pkinase 19 273 359.7  4.10E−105 76 159 NAF 324 381 105.6 1.30E−28 77160 SRF-TF 9 59 100.8 3.60E−27 77 160 K-box 73 173 95.3 1.60E−25 50 133Peptidase_S10 1 227 −42.7 6.00E−11 51 134 Ank 44 76 47.3 4.70E−11 51 134Ank 77 109 33.5 6.40E−07 51 134 Ank 111 144 15.7 0.14  51 134 Ank 185217 39.7 9.00E−09 51 134 Ank 228 260 30.7 4.50E−06 52 135 Pkinase_Tyr 51341 158.7 1.40E−44 52 135 Pkinase 63 341 104.4 3.00E−28 54 137 GATase_22 162 11.8 6.10E−12 54 137 Asn_synthase 211 479 334.3 1.80E−97 55 138HSP20 56 164 168.2 1.90E−47 78 161 Lactamase_B 93 251 129 1.20E−35 56139 UPF0057 11 62 102.9 8.40E−28 57 140 Oxidored_FMN 6 341 302.19.10E−88 58 141 Pkinase 39 325 309.2 6.40E−90 59 142 Pyridoxal_deC 33381 546  3.40E−161 60 143 Pyridoxal_deC 33 381 546  3.40E−161 61 144HSP20 57 160 178.8 1.20E−50 38 121 PDZ 200 284 37.6 3.80E−08 38 121Peptidase_S41 320 483 244.5 1.90E−70 62 145 Cpn60_TCP1 59 562 578.6 5.40E−171 63 146 DSPc 50 188 142.9 7.70E−40 64 147 Isoamylase_N 61 14994.9 2.10E−25 64 147 Alpha-amylase 209 589 −36.4 1.30E−07 79 162 Pkinase45 299 360.3  2.80E−105 79 162 NAF 384 441 105.2 1.70E−28 65 148 DUF168538 146 184.5 2.40E−52 80 163 GAF 219 404 108.4 1.90E−29 80 163Phytochrome 415 595 409.1  5.70E−120 80 163 PAS 622 737 96.6 6.50E−26 80163 PAS 752 877 107.4 3.80E−29 80 163 HisKA 897 956 26.7 7.10E−05 80 163HATPase_c 1011 1123 64.4 3.30E−16 66 149 Glyco_hydro_1 74 558 1024.90    67 150 ArfGap 17 133 174.4 2.50E−49 81 164 AP2 6 69 132 1.50E−36

TABLE 27 accession gathering pfam domain name number cutoff domaindescription AAA PF00004.17 10 ATPase family associated with variouscellular activities (AAA) AP2 PF00847.9 0 AP2 domain Aldo_ket_redPF00248.10 −97 Aldo/keto reductase family Alpha-amylase PF00128.11 −93Alpha amylase, catalytic domain Aminotran_1_2 PF00155.9 −57.5Aminotransferase class I and II Ank PF00023.17 21.6 Ankyrin repeatArfGap PF01412.8 −17 Putative GTPase activating protein for ArfAsn_synthase PF00733.10 −52.8 Asparagine synthase BRO1 PF03097.6 25BRO1-like domain CBFD_NFYB_HMF PF00808.12 18.4 Histone-liketranscription factor (CBF/NF- Y) and archaeal histone Catalase PF00199.8−229 Catalase CorA PF01544.8 −61.3 CorA-like Mg2+ transporter proteinCpn60_TCP1 PF00118.13 −223.4 TCP-1/cpn60 chaperonin family CystatinPF00031.10 17.5 Cystatin domain DNA_photolyase PF00875.7 −10 DNAphotolyase DSPc PF00782.9 −21.8 Dual specificity phosphatase, catalyticdomain DUF1685 PF07939.1 25 Protein of unknown function (DUF1685) DUF296PF03479.4 −11 Domain of unknown function (DUF296) Di19 PF05605.2 25Drought induced 19 protein (Di19) E2F_TOP PF02319.9 17 E2F/DP familywinged-helix DNA- binding domain FAD_binding_7 PF03441.3 25 FAD bindingdomain of DNA photolyase FA_desaturase PF00487.13 −46 Fatty aciddesaturase FBPase PF00316.9 −170.3 Fructose-1-6-bisphosphatase GAFPF01590.14 23 GAF domain GATA PF00320.15 28.5 GATA zinc finger GATase_2PF00310.10 −106.2 Glutamine amidotransferases class-II Glyco_hydro_1PF00232.8 −301.8 Glycosyl hydrolase family 1 Glyoxalase PF00903.14 12.1Glyoxalase/Bleomycin resistance protein/Dioxygenase superfamily Got1PF04178.2 25 Got1-like family HATPase_c PF02518.13 22.4 Histidinekinase-, DNA gyrase B-, and HSP90-like ATPase HSF_DNA-bind PF00447.7 −70HSF-type DNA-binding HSP20 PF00011.9 13 Hsp20/alpha crystallin familyHisKA PF00512.13 10.2 His Kinase A (phosphoacceptor) domain HomeoboxPF00046.17 −4.1 Homeobox domain Hpt PF01627.11 25 Hpt domainIsoamylase_N PF02922.7 −6.5 Isoamylase N-terminal domain K-box PF01486.70 K-box region Lactamase_B PF00753.15 22.3 Metallo-beta-lactamasesuperfamily Metallophos PF00149.16 22 Calcineurin-like phosphoesteraseMtN3_slv PF03083.5 −0.8 MtN3/saliva family NAF PF03822.4 25 NAF domainNAM PF02365.5 −19 No apical meristem (NAM) protein NIF PF03031.7 −81 NLIinteracting factor-like phosphatase Oxidored_FMN PF00724.8 −147.7 NADH:flavin oxidoreductase/NADH oxidase family PAS PF00989.12 20 PAS fold PDZPF00595.11 12.1 PDZ domain (Also known as DHR or GLGF) PRA1 PF03208.8 25PRA1 family protein Peptidase_C15 PF01470.7 −100 Pyroglutamyl peptidasePeptidase_S10 PF00450.11 −198 Serine carboxypeptidase Peptidase_S41PF03572.7 −25.8 Peptidase family S41 Phytochrome PF00360.9 11Phytochrome region Pkinase PF00069.14 −70.8 Protein kinase domainPkinase_Tyr PF07714.4 65 Protein tyrosine kinase Pyridoxal_deC PF00282.8−158.6 Pyridoxal-dependent decarboxylase conserved domain RIO1PF01163.11 −89.1 RIO1 family RRM_1 PF00076.10 15.2 RNA recognitionmotif, (a.k.a. RRM, RBD, or RNP domain) RTC PF01137.11 −36.9 RNA3′-terminal phosphate cyclase RTC_insert PF05189.3 25 RNA 3′-terminalphosphate cyclase (RTC), insert domain Ras PF00071.11 18 Ras familyResponse_reg PF00072.11 −14.4 Response regulator receiver domain SPC25PF06703.1 25 Microsomal signal peptidase 25 kDa subunit (SPC25) SPXPF03105.9 −20 SPX domain SRF-TF PF00319.8 11 SRF-type transcriptionfactor (DNA- binding and dimerisation domain) Synaptobrevin PF00957.9 25Synaptobrevin UPF0057 PF01679.7 25 Uncharacterized protein familyUPF0057 zf-C2H2 PF00096.14 19 Zinc finger, C2H2 type zf-C3HC4 PF00097.1216.9 Zinc finger, C3HC4 type (RING finger)

Example 8 Selection of Transgenic Plants with Enhanced AgronomicTrait(s)

This example illustrates the preparation and identification by selectionof transgenic seeds and plants derived from transgenic plant cells ofthis invention where the plants and seed are identified by screening ahaving an enhanced agronomic trait imparted by expression of a proteinselected from the group including the homologous proteins identified inExample 4, SEQ ID NO: 121, 128, 152-160, 162 and 164. Transgenic plantcells of corn, soybean, cotton, canola, wheat and rice are transformedwith recombinant DNA for expressing each of the homologs identified inExample 4. Plants are regenerated from the transformed plant cells andused to produce progeny plants and seed that are screened for enhancedwater use efficiency, enhanced cold tolerance, increased yield, enhancednitrogen use efficiency, enhanced seed protein and enhanced seed oil.Plants are identified exhibiting enhanced traits imparted by expressionof the homologous proteins.

What is claimed is:
 1. A plant cell with stably integrated, recombinantDNA comprising a promoter that is functional in plant cells and that isoperably linked to DNA from a plant, bacteria or yeast that encodes aprotein having at least one domain of amino acids in a sequence thatexceeds the Pfam gathering cutoff for amino acid sequence alignment witha protein domain family identified by a Pfam name in the group of Pfamnames consisting of AAA, AP2, Aldo ket red, Alpha-amylase, Aminotran 12, Ank, ArfGap, Asn synthase, BRO1, CBFD NFYB HMF, Catalase, CorA, Cpn60TCP 1, Cystatin, DNA photolyase, DSPc, DUF1685, DUF296, Di19, E2F TDP,FAD binding 7, FA desaturase, FBPase, GAF, GATA, GATase 2, Glyco hydro1, Glyoxalase, Got1, HATPase c, HSF DNA-bind, HSP20, HisKA, Homeobox,Hpt, Isoamylase N, K-box, Lactamase B, Metallophos, MtN3 slv, NAF, NAM,NIF, Oxidored FMN, PAS, PDZ, PRA1, Peptidase C15, Peptidase S10,Peptidase S41, Phytochrome, Peinase, Pkinase Tyr, Pyridoxal deC, RIO1,RRM 1, RTC, RTC insert, Ras, Response reg, SPC25, SPX, SRF-TF,Synaptobrevin, UPF0057, zf-C2H2, and zf-C3HC4; wherein the Pfamgathering cuttoff for said protein domain families is stated in Table28; wherein said plant cell is selected from a population of plant cellswith said recombinant DNA by screening plants that are regenerated fromplant cells in said population and that express said protein for anenhanced trait as compared to control plants that do not have saidrecombinant DNA; and wherein said enhanced trait is selected from groupof enhanced traits consisting of enhanced water use efficiency, enhancedcold tolerance, increased yield, enhanced nitrogen use efficiency,enhanced seed protein and enhanced seed oil.
 2. A plant cell of claim 1wherein said protein has an amino acid sequence with at least 90%identity to a consensus amino acid sequence in the group of consensusamino acid sequences consisting of the consensus amino acid sequenceconstructed for SEQ ID NO:84 and homologs thereof listed in Table 2through the consensus amino acid sequence constructed for SEQ ID NO:166and homologs thereof listed in Table
 2. 3. A plant cell of claim 1wherein said protein is selected from the group of proteins identifiedin Table
 1. 4. A plant cell of claim 1 further comprising DNA expressinga protein that provides tolerance from exposure to an herbicide appliedat levels that are lethal to a wild type of said plant cell.
 5. A plantcell of claim 4 wherein the agent of said herbicide is a glyphosate,dicamba, or glufosinate compound.
 6. A transgenic plant comprising aplurality of the plant cell of claim
 1. 7. A transgenic plant of claim 6which is homozygous for said recombinant DNA.
 8. A transgenic seedcomprising a plurality of the plant cell of claim
 1. 9. A transgenicseed of claim 8 from a corn, soybean, cotton, canola, alfalfa, wheat orrice plant.
 10. Non-natural, transgenic corn seed of claim 9 whereinsaid seed can produce corn plants that are resistant to disease from theMal de Rio Cuarto virus or the Puccina sorghi fungus or both.
 11. Atransgenic pollen grain comprising a haploid derivative of a plant cellof claim
 1. 12. A method for manufacturing non-natural, transgenic seedthat can be used to produce a crop of transgenic plants with an enhancedtrait resulting from expression of stably-integrated, recombinant DNAcomprising a promoter that is (a) functional in plant cells and (b) isoperably linked to DNA from a plant, bacteria or yeast that encodes aprotein having at least one domain of amino acids in a sequence thatexceeds the Pfam gathering cutoff for amino acid sequence alignment witha protein domain family identified by a Pfam name in the group of Pfamnames consisting of AAA, AP2, Aldo ket red, Alpha-amylase, Aminotran 12, Ank, ArfGap, Asn synthase, BRO1, CBFD NFYB HMF, Catalase, CorA, Cpn60TCP1, Cystatin, DNA photolyase, DSPc, DUF1685, DUF296, Di19, E2F TDP,FAD binding 7, FA desaturase, FBPase, GAF, GATA, GATase 2, Glyco hydro1, Glyoxalase, Got1, HATPase c, HSF DNA-bind, HSP20, HisKA, Homeobox,Hpt, Isoamylase N, K-box, Lactamase B, Metallophos, MtN3 slv, NAF, NAM,NIF, Oxidored FMN, PAS, PDZ, PRA1, Peptidase C15, Peptidase S10,Peptidase S41, Phytochrome, Peinase, Pkinase Tyr, Pyridoxal deC, RIO1,RRM 1, RTC, RTC insert, Ras, Response reg, SPC25, SPX, SRF-TF,Synaptobrevin, UPF0057, zf-C2H2, and zf-C3HC4; wherein the gatheringcutoff for said protein domain families is stated in Table 28; andwherein said enhanced trait is selected from the group of enhancedtraits consisting of enhanced water use efficiency, enhanced coldtolerance, increased yield, enhanced nitrogen use efficiency, enhancedseed protein and enhanced seed oil, said method for manufacturing saidseed comprising: (a) screening a population of plants for said enhancedtrait and said recombinant DNA, wherein individual plants in saidpopulation can exhibit said trait at a level less than, essentially thesame as or greater than the level that said trait is exhibited incontrol plants which do not express the recombinant DNA, (b) selectingfrom said population one or more plants that exhibit the trait at alevel greater than the level that said trait is exhibited in controlplants, (c) verifying that said recombinant DNA is stably integrated insaid selected plants, (d) analyzing tissue of a selected plant todetermine the production of a protein having the function of a proteinencoded by nucleotides in a sequence of one of SEQ ID NO:1-83; and (e)collecting seed from a selected plant.
 13. A method of claim 12 whereinplants in said population further comprise DNA expressing a protein thatprovides tolerance to exposure to an herbicide applied at levels thatare lethal to wild type plant cells, and wherein said selecting iseffected by treating said population with said herbicide.
 14. A methodof claim 13 wherein said herbicide comprises a glyphosate, dicamba, orglufosinate compound.
 15. A method of claim 12 wherein said selecting iseffected by identifying plants with said enhanced trait.
 16. A method ofclaim 12 wherein said seed is corn, soybean, cotton, alfalfa, wheat orrice seed.
 17. A method of producing hybrid corn seed comprising: (a)acquiring hybrid corn seed from a herbicide tolerant corn plant whichalso has stably-integrated, recombinant DNA comprising a promoter thatis (a) functional in plant cells and (b) is operably linked to DNA thatencodes a protein having at least one domain of amino acids in asequence that exceeds the Pfam gathering cutoff for amino acid sequencealignment with a protein domain family identified by a Pfam name in thegroup of Pfam names consisting of AAA, AP2, Aldo ket red, Alpha-amylase,Aminotran 1 2, Ank, ArfGap, Asn synthase, BRO1, CBFD NFYB HMF, Catalase,CorA, Cpn60 TCP1, Cystatin, DNA photolyase, DSPc, DUF1685, DUF296, Di19,E2F TDP, FAD binding 7, FA desaturase, FBPase, GAF, GATA, GATase 2,Glyco hydro 1, Glyoxalase, Got1, HATPase c, HSF DNA-bind, HSP20, HisKA,Homeobox, Hpt, Isoamylase N, K-box, Lactamase B, Metallophos, MtN3 slv,NAF, NAM, NIF, Oxidored FMN, PAS, PDZ, PRA1, Peptidase C15, PeptidaseS10, Peptidase S41, Phytochrome, Peinase, Pkinase Tyr, Pyridoxal deC,RIO1, RRM 1, RTC, RTC insert, Ras, Response reg, SPC25, SPX, SRF-TF,Synaptobrevin, UPF0057, zf-C2H2, and zf-C3HC4; wherein the gatheringcuttoff for said protein domain families is stated in Table 28; (b)producing corn plants from said hybrid corn seed, wherein a fraction ofthe plants produced from said hybrid corn seed is homozygous for saidrecombinant DNA, a fraction of the plants produced from said hybrid cornseed is hemizygous for said recombinant DNA, and a fraction of theplants produced from said hybrid corn seed has none of said recombinantDNA; (c) selecting corn plants which are homozygous and hemizygous forsaid recombinant DNA by treating with an herbicide; (d) collecting seedfrom herbicide-treated-surviving corn plants and planting said seed toproduce further progeny corn plants; (e) repeating steps (c) and (d) atleast once to produce an inbred corn line; (f) crossing said inbred cornline with a second corn line to produce hybrid seed.
 18. The method ofselecting a plant comprising cells of claim 1 wherein an immunoreactiveantibody is used to detect the presence of said protein in seed or planttissue.
 19. Anti-counterfeit milled seed having, as an indication oforigin, a plant cell of claim
 1. 20. A method of growing a corn, cottonor soybean crop without irrigation water comprising planting seed havingplant cells of claim 1 which are selected for enhanced water useefficiency.
 21. A method of claim 20 comprising providing up to 300millimeters of ground water during the production of said crop.
 22. Amethod of growing a corn, cotton or soybean crop without added nitrogenfertilizer comprising planting seed having plant cells of claim 1 whichare selected for enhanced nitrogen use efficiency.